diff --git a/.changeset/rich-pans-press.md b/.changeset/rich-pans-press.md
new file mode 100644
index 00000000000..15ab412aa26
--- /dev/null
+++ b/.changeset/rich-pans-press.md
@@ -0,0 +1,5 @@
+---
+"@primer/react": patch
+---
+
+TreeView: Always align expand/collapse chevron icon, leading visual, and trailing visual to top of item
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index 9f8888445bf..a9cc60d52b3 100644
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diff --git a/.playwright/snapshots/components/TreeView.test.ts-snapshots/TreeView-Leading-Action-light-tritanopia-linux.png b/.playwright/snapshots/components/TreeView.test.ts-snapshots/TreeView-Leading-Action-light-tritanopia-linux.png
index e5428c1f3a9..febee9c17e5 100644
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diff --git a/packages/react/src/TreeView/TreeView.features.stories.tsx b/packages/react/src/TreeView/TreeView.features.stories.tsx
index 73d02f13d75..639fd6602e7 100644
--- a/packages/react/src/TreeView/TreeView.features.stories.tsx
+++ b/packages/react/src/TreeView/TreeView.features.stories.tsx
@@ -1040,4 +1040,88 @@ export const LeadingAction: Story = () => {
   )
 }
 
+export const MultilineItems: Story = () => (
+  <nav aria-label="Files changed">
+    <TreeView aria-label="Files changed">
+      <TreeView.Item id="src" defaultExpanded>
+        <TreeView.LeadingVisual>
+          <TreeView.DirectoryIcon />
+        </TreeView.LeadingVisual>
+        <div style={{whiteSpace: 'wrap'}}>
+          this is a very long directory name that we have intentionally allowed to wrap over multiple lines to
+          demonstrate alignment
+        </div>
+        <TreeView.SubTree>
+          <TreeView.Item id="src/Avatar.tsx">
+            <TreeView.LeadingVisual>
+              <FileIcon />
+            </TreeView.LeadingVisual>
+            Avatar.tsx
+            <TreeView.TrailingVisual>
+              <Octicon icon={DiffAddedIcon} color="success.fg" aria-label="Added" />
+            </TreeView.TrailingVisual>
+          </TreeView.Item>
+        </TreeView.SubTree>
+      </TreeView.Item>
+      <TreeView.Item id="src" defaultExpanded>
+        <TreeView.LeadingVisual>
+          <TreeView.DirectoryIcon />
+        </TreeView.LeadingVisual>
+        <div style={{whiteSpace: 'wrap'}}>
+          this is a medium directory name that we wrap over 2 lines to demonstrate alignment
+        </div>
+        <TreeView.TrailingVisual>
+          <Octicon icon={DiffAddedIcon} color="success.fg" aria-label="Added" />
+        </TreeView.TrailingVisual>
+        <TreeView.SubTree>
+          <TreeView.Item id="src/Avatar.tsx">
+            <TreeView.LeadingVisual>
+              <FileIcon />
+            </TreeView.LeadingVisual>
+            Avatar.tsx
+            <TreeView.TrailingVisual>
+              <Octicon icon={DiffAddedIcon} color="success.fg" aria-label="Added" />
+            </TreeView.TrailingVisual>
+          </TreeView.Item>
+        </TreeView.SubTree>
+      </TreeView.Item>
+      <TreeView.Item id="src" defaultExpanded>
+        <TreeView.LeadingVisual>
+          <TreeView.DirectoryIcon />
+        </TreeView.LeadingVisual>
+        this is a very long directory name that we have intentionally NOT allowed to wrap over multiple lines to
+        demonstrate alignment
+        <TreeView.SubTree>
+          <TreeView.Item id="src/Avatar.tsx">
+            <TreeView.LeadingVisual>
+              <FileIcon />
+            </TreeView.LeadingVisual>
+            Avatar.tsx
+            <TreeView.TrailingVisual>
+              <Octicon icon={DiffAddedIcon} color="success.fg" aria-label="Added" />
+            </TreeView.TrailingVisual>
+          </TreeView.Item>
+        </TreeView.SubTree>
+      </TreeView.Item>
+      <TreeView.Item id="src" defaultExpanded>
+        <TreeView.LeadingVisual>
+          <TreeView.DirectoryIcon />
+        </TreeView.LeadingVisual>
+        short name
+        <TreeView.SubTree>
+          <TreeView.Item id="src/Avatar.tsx">
+            <TreeView.LeadingVisual>
+              <FileIcon />
+            </TreeView.LeadingVisual>
+            Avatar.tsx
+            <TreeView.TrailingVisual>
+              <Octicon icon={DiffAddedIcon} color="success.fg" aria-label="Added" />
+            </TreeView.TrailingVisual>
+          </TreeView.Item>
+        </TreeView.SubTree>
+      </TreeView.Item>
+    </TreeView>
+  </nav>
+)
+
 export default meta
diff --git a/packages/react/src/TreeView/TreeView.tsx b/packages/react/src/TreeView/TreeView.tsx
index 53e86b916f5..06aa13321c6 100644
--- a/packages/react/src/TreeView/TreeView.tsx
+++ b/packages/react/src/TreeView/TreeView.tsx
@@ -67,6 +67,9 @@ export type TreeViewProps = {
   className?: string
 }
 
+/* Size of toggle icon in pixels. */
+const TOGGLE_ICON_SIZE = 12
+
 const UlBox = styled.ul<SxProp>`
   list-style: none;
   padding: 0;
@@ -105,6 +108,7 @@ const UlBox = styled.ul<SxProp>`
   .PRIVATE_TreeView-item-container {
     --level: 1; /* default level */
     --toggle-width: 1rem; /* 16px */
+    --min-item-height: 2rem; /* 32px */
     position: relative;
     display: grid;
     --leading-action-width: calc(var(--has-leading-action, 0) * 1.5rem);
@@ -112,7 +116,6 @@ const UlBox = styled.ul<SxProp>`
     grid-template-columns: var(--spacer-width) var(--leading-action-width) var(--toggle-width) 1fr;
     grid-template-areas: 'spacer leadingAction toggle content';
     width: 100%;
-    min-height: 2rem; /* 32px */
     font-size: ${get('fontSizes.1')};
     color: ${get('colors.fg.default')};
     border-radius: ${get('radii.2')};
@@ -129,7 +132,7 @@ const UlBox = styled.ul<SxProp>`
 
     @media (pointer: coarse) {
       --toggle-width: 1.5rem; /* 24px */
-      min-height: 2.75rem; /* 44px */
+      --min-item-height: 2.75rem; /* 44px */
     }
 
     &:has(.PRIVATE_TreeView-item-skeleton):hover {
@@ -169,8 +172,11 @@ const UlBox = styled.ul<SxProp>`
   .PRIVATE_TreeView-item-toggle {
     grid-area: toggle;
     display: flex;
-    align-items: center;
     justify-content: center;
+    align-items: flex-start;
+    /* The toggle should appear vertically centered for single-line items, but remain at the top for items that wrap
+    across more lines. */
+    padding-top: calc(var(--min-item-height) / 2 - ${TOGGLE_ICON_SIZE}px / 2);
     height: 100%;
     color: ${get('colors.fg.muted')};
   }
@@ -187,10 +193,13 @@ const UlBox = styled.ul<SxProp>`
   .PRIVATE_TreeView-item-content {
     grid-area: content;
     display: flex;
-    align-items: center;
     height: 100%;
     padding: 0 ${get('space.2')};
     gap: ${get('space.2')};
+    line-height: var(--custom-line-height, var(--text-body-lineHeight-medium, 1.4285));
+    /* The dynamic top and bottom padding to maintain the minimum item height for single line items */
+    padding-top: calc((var(--min-item-height) - var(--custom-line-height, 1.3rem)) / 2);
+    padding-bottom: calc((var(--min-item-height) - var(--custom-line-height, 1.3rem)) / 2);
   }
 
   .PRIVATE_TreeView-item-content-text {
@@ -204,7 +213,11 @@ const UlBox = styled.ul<SxProp>`
 
   .PRIVATE_TreeView-item-visual {
     display: flex;
+    align-items: center;
     color: ${get('colors.fg.muted')};
+    /* The visual icons should appear vertically centered for single-line items, but remain at the top for items that wrap
+    across more lines. */
+    height: var(--custom-line-height, 1.3rem);
   }
 
   .PRIVATE_TreeView-item-leading-action {
@@ -524,7 +537,11 @@ const Item = React.forwardRef<HTMLElement, TreeViewItemProps>(
                   }
                 }}
               >
-                {isExpanded ? <ChevronDownIcon size={12} /> : <ChevronRightIcon size={12} />}
+                {isExpanded ? (
+                  <ChevronDownIcon size={TOGGLE_ICON_SIZE} />
+                ) : (
+                  <ChevronRightIcon size={TOGGLE_ICON_SIZE} />
+                )}
               </div>
             ) : null}
             <div id={labelId} className="PRIVATE_TreeView-item-content">