diff --git a/mypy/semanal.py b/mypy/semanal.py
index 11f0156372bf..560d4eeb74e9 100644
--- a/mypy/semanal.py
+++ b/mypy/semanal.py
@@ -4014,12 +4014,13 @@ def analyze_alias(
                 python_3_12_type_alias=python_3_12_type_alias,
             )
 
-        # There can be only one variadic variable at most, the error is reported elsewhere.
+        # Check for multiple TypeVarTuples in type alias definition
         new_tvar_defs = []
         variadic = False
         for td in tvar_defs:
             if isinstance(td, TypeVarTupleType):
                 if variadic:
+                    self.fail("Can only use one TypeVarTuple in a type alias", rvalue)
                     continue
                 variadic = True
             new_tvar_defs.append(td)
diff --git a/test-data/unit/check-python312.test b/test-data/unit/check-python312.test
index 0521722b47c1..b09693091d1f 100644
--- a/test-data/unit/check-python312.test
+++ b/test-data/unit/check-python312.test
@@ -2237,3 +2237,26 @@ class D[*Ts](Generic[Unpack[Us]]):  # E: Generic[...] base class is redundant \
                                     # E: Can only use one type var tuple in a class def
     pass
 [builtins fixtures/tuple.pyi]
+
+[case testPEP695MultipleTypeVarTuplesTypeAlias]
+type TA1[*Ts1, *Ts2] = tuple[*Ts1] | tuple[*Ts2]  # E: Can only use one TypeVarTuple in a type alias
+type TA2[T, *Ts1, *Ts2] = tuple[T, *Ts1, *Ts2]  # E: Can only use one TypeVarTuple in a type alias \
+                                                 # E: More than one variadic Unpack in a type is not allowed
+type TA3[*Ts1, T, *Ts2] = tuple[*Ts1, T, *Ts2]  # E: Can only use one TypeVarTuple in a type alias \
+                                                 # E: More than one variadic Unpack in a type is not allowed
+type TA4[*Ts] = tuple[*Ts]  # OK - single TypeVarTuple is fine
+[builtins fixtures/tuple.pyi]
+[typing fixtures/typing-full.pyi]
+
+
+[case testMultipleTypeVarTuplesOldStyle]
+# flags: --python-version 3.12
+from typing import Union
+from typing_extensions import TypeVarTuple, Unpack
+
+Ts1 = TypeVarTuple("Ts1")
+Ts2 = TypeVarTuple("Ts2")
+
+TA = Union[tuple[*Ts1], tuple[*Ts2]]  # E: Can only use one TypeVarTuple in a type alias
+[builtins fixtures/tuple.pyi]
+[typing fixtures/typing-full.pyi]
diff --git a/test-data/unit/check-typevar-tuple.test b/test-data/unit/check-typevar-tuple.test
index f6d9de8b0c1a..f9192560cf52 100644
--- a/test-data/unit/check-typevar-tuple.test
+++ b/test-data/unit/check-typevar-tuple.test
@@ -725,15 +725,18 @@ Ts = TypeVarTuple("Ts")
 Us = TypeVarTuple("Us")
 class G(Generic[Unpack[Ts]]): ...
 
-A = Tuple[Unpack[Ts], Unpack[Us]]  # E: More than one variadic Unpack in a type is not allowed
+A = Tuple[Unpack[Ts], Unpack[Us]]  # E: Can only use one TypeVarTuple in a type alias \
+                                   # E: More than one variadic Unpack in a type is not allowed
 x: A[int, str]
 reveal_type(x)  # N: Revealed type is "tuple[builtins.int, builtins.str]"
 
-B = Callable[[Unpack[Ts], Unpack[Us]], int]  # E: More than one variadic Unpack in a type is not allowed
+B = Callable[[Unpack[Ts], Unpack[Us]], int]  # E: Can only use one TypeVarTuple in a type alias \
+                                             # E: More than one variadic Unpack in a type is not allowed
 y: B[int, str]
 reveal_type(y)  # N: Revealed type is "def (builtins.int, builtins.str) -> builtins.int"
 
-C = G[Unpack[Ts], Unpack[Us]]  # E: More than one variadic Unpack in a type is not allowed
+C = G[Unpack[Ts], Unpack[Us]]  # E: Can only use one TypeVarTuple in a type alias \
+                                  # E: More than one variadic Unpack in a type is not allowed
 z: C[int, str]
 reveal_type(z)  # N: Revealed type is "__main__.G[builtins.int, builtins.str]"
 [builtins fixtures/tuple.pyi]