diff --git a/brainglobe_atlasapi/atlas_generation/validate_atlases.py b/brainglobe_atlasapi/atlas_generation/validate_atlases.py
index e8524b59..230944fb 100644
--- a/brainglobe_atlasapi/atlas_generation/validate_atlases.py
+++ b/brainglobe_atlasapi/atlas_generation/validate_atlases.py
@@ -424,6 +424,45 @@ def validate_annotation_symmetry(atlas: BrainGlobeAtlas):
     return True
 
 
+def validate_unique_acronyms(atlas: BrainGlobeAtlas):
+    """Validate that all structure acronyms in the atlas are unique.
+
+    Duplicate acronyms are incompatible with the current implementation
+    of brainglobe-atlasapi as the acronym is used as a primary key to
+    fetch details for a region.
+
+    Parameters
+    ----------
+    atlas : BrainGlobeAtlas
+        The BrainGlobeAtlas object to validate.
+
+    Returns
+    -------
+    bool
+        True if all acronyms are unique.
+
+    Raises
+    ------
+    AssertionError
+        If any duplicate acronyms are found in the atlas structures.
+    """
+    seen = set()
+    duplicates = []
+
+    for structure in atlas.structures:
+        acronym = atlas.structures[structure]["acronym"]
+        if acronym in seen:
+            name = atlas.structures[structure]["name"]
+            duplicates.append((acronym, name))
+        else:
+            seen.add(acronym)
+
+    assert (
+        len(duplicates) == 0
+    ), f"Duplicate acronyms found in atlas structures: {sorted(duplicates)}"
+    return True
+
+
 def validate_atlas_name(atlas: BrainGlobeAtlas):
     """Validate the naming convention of the atlas.
 
@@ -523,6 +562,7 @@ def get_all_validation_functions():
         catch_missing_structures,
         validate_reference_image_pixels,
         validate_annotation_symmetry,
+        validate_unique_acronyms,
         validate_atlas_name,
     ]
 
@@ -588,6 +628,7 @@ def validate_atlas(atlas_name, version, validation_functions):
         catch_missing_structures,
         validate_reference_image_pixels,
         validate_annotation_symmetry,
+        validate_unique_acronyms,
         validate_atlas_name,
     ]
 
diff --git a/tests/atlasgen/test_validation.py b/tests/atlasgen/test_validation.py
index 232cfaa5..b06bfb97 100644
--- a/tests/atlasgen/test_validation.py
+++ b/tests/atlasgen/test_validation.py
@@ -21,6 +21,7 @@
     validate_mesh_matches_image_extents,
     validate_metadata,
     validate_reference_image_pixels,
+    validate_unique_acronyms,
 )
 from brainglobe_atlasapi.config import get_brainglobe_dir
 from brainglobe_atlasapi.core import AdditionalRefDict
@@ -457,3 +458,31 @@ def test_validate_metadata(atlas, metadata, expected_output, error_message):
             validate_metadata(atlas)
     else:
         assert validate_metadata(atlas) == expected_output
+
+
+def test_validate_unique_acronyms_fail(mocker, atlas):
+    """Check that an atlas with duplicate acronyms fails validation.
+
+    Parameters
+    ----------
+    mocker : pytest_mock.MockerFixture
+        Mocker fixture for patching.
+    atlas : BrainGlobeAtlas
+        A BrainGlobeAtlas instance.
+    """
+    # Create structures with duplicate acronyms
+    structures_with_duplicates = {
+        1: {"acronym": "root", "name": "Root"},
+        2: {"acronym": "brain", "name": "Brain"},
+        3: {"acronym": "brain", "name": "Brain Duplicate"},  # Duplicate!
+        4: {"acronym": "cortex", "name": "Cortex"},
+    }
+    mocker.patch.object(atlas, "structures", structures_with_duplicates)
+
+    with pytest.raises(AssertionError) as exc_info:
+        validate_unique_acronyms(atlas)
+
+    # Verify error contains the duplicate acronym and its name
+    error_message = str(exc_info.value)
+    assert "brain" in error_message
+    assert "Brain Duplicate" in error_message