diff --git a/doc/whats-new.rst b/doc/whats-new.rst
index 6db780484bd..31d3a1077e7 100644
--- a/doc/whats-new.rst
+++ b/doc/whats-new.rst
@@ -34,6 +34,8 @@ Bug fixes
   By `Kai Mühlbauer <https://github.com/kmuehlbauer>`_.
 - Fix the error message of :py:func:`testing.assert_equal` when two different :py:class:`DataTree` objects
   are passed (:pull:`10440`). By `Mathias Hauser <https://github.com/mathause>`_.
+- Fix :py:func:`testing.assert_equal` with ``check_dim_order=False`` for :py:class:`DataTree` objects
+  (:pull:`10442`). By `Mathias Hauser <https://github.com/mathause>`_.
 
 
 Documentation
diff --git a/xarray/testing/assertions.py b/xarray/testing/assertions.py
index e524603c9a5..474a72da739 100644
--- a/xarray/testing/assertions.py
+++ b/xarray/testing/assertions.py
@@ -12,6 +12,7 @@
 from xarray.core.dataarray import DataArray
 from xarray.core.dataset import Dataset
 from xarray.core.datatree import DataTree
+from xarray.core.datatree_mapping import map_over_datasets
 from xarray.core.formatting import diff_datatree_repr
 from xarray.core.indexes import Index, PandasIndex, PandasMultiIndex, default_indexes
 from xarray.core.variable import IndexVariable, Variable
@@ -85,14 +86,25 @@ def assert_isomorphic(a: DataTree, b: DataTree):
 
 def maybe_transpose_dims(a, b, check_dim_order: bool):
     """Helper for assert_equal/allclose/identical"""
+
     __tracebackhide__ = True
-    if not isinstance(a, Variable | DataArray | Dataset):
+
+    def _maybe_transpose_dims(a, b):
+        if not isinstance(a, Variable | DataArray | Dataset):
+            return b
+        if set(a.dims) == set(b.dims):
+            # Ensure transpose won't fail if a dimension is missing
+            # If this is the case, the difference will be caught by the caller
+            return b.transpose(*a.dims)
+        return b
+
+    if check_dim_order:
         return b
-    if not check_dim_order and set(a.dims) == set(b.dims):
-        # Ensure transpose won't fail if a dimension is missing
-        # If this is the case, the difference will be caught by the caller
-        return b.transpose(*a.dims)
-    return b
+
+    if isinstance(a, DataTree):
+        return map_over_datasets(_maybe_transpose_dims, a, b)
+
+    return _maybe_transpose_dims(a, b)
 
 
 @ensure_warnings
diff --git a/xarray/tests/test_assertions.py b/xarray/tests/test_assertions.py
index cef965f9854..a0a2c02d578 100644
--- a/xarray/tests/test_assertions.py
+++ b/xarray/tests/test_assertions.py
@@ -88,6 +88,19 @@ def test_assert_allclose_equal_transpose(func) -> None:
     getattr(xr.testing, func)(ds1, ds2, check_dim_order=False)
 
 
+def test_assert_equal_transpose_datatree() -> None:
+    """Ensure `check_dim_order=False` works for transposed DataTree"""
+    ds = xr.Dataset(data_vars={"data": (("x", "y"), [[1, 2]])})
+
+    a = xr.DataTree.from_dict({"node": ds})
+    b = xr.DataTree.from_dict({"node": ds.transpose("y", "x")})
+
+    with pytest.raises(AssertionError):
+        xr.testing.assert_equal(a, b)
+
+    xr.testing.assert_equal(a, b, check_dim_order=False)
+
+
 @pytest.mark.filterwarnings("error")
 @pytest.mark.parametrize(
     "duckarray",