diff --git a/doc/source/whatsnew/v1.4.1.rst b/doc/source/whatsnew/v1.4.1.rst
index 21aab5058d25b..d0a58a19df92f 100644
--- a/doc/source/whatsnew/v1.4.1.rst
+++ b/doc/source/whatsnew/v1.4.1.rst
@@ -15,6 +15,7 @@ including other versions of pandas.
 Fixed regressions
 ~~~~~~~~~~~~~~~~~
 - Regression in :meth:`Series.mask` with ``inplace=True`` and ``PeriodDtype`` and an incompatible ``other`` coercing to a common dtype instead of raising (:issue:`45546`)
+- Regression in :func:`.assert_frame_equal` not respecting ``check_flags=False`` (:issue:`45554`)
 - Regression in :meth:`Series.fillna` with ``downcast=False`` incorrectly downcasting ``object`` dtype (:issue:`45603`)
 - Regression in :meth:`DataFrame.loc.__setitem__` losing :class:`Index` name if :class:`DataFrame` was empty before (:issue:`45621`)
 -
diff --git a/pandas/_testing/asserters.py b/pandas/_testing/asserters.py
index ea75af20bb0b6..77e4065008804 100644
--- a/pandas/_testing/asserters.py
+++ b/pandas/_testing/asserters.py
@@ -1344,6 +1344,7 @@ def assert_frame_equal(
                 rtol=rtol,
                 atol=atol,
                 check_index=False,
+                check_flags=False,
             )
 
 
diff --git a/pandas/tests/util/test_assert_frame_equal.py b/pandas/tests/util/test_assert_frame_equal.py
index faea0a54dc330..6ff1a1c17b179 100644
--- a/pandas/tests/util/test_assert_frame_equal.py
+++ b/pandas/tests/util/test_assert_frame_equal.py
@@ -323,9 +323,22 @@ def test_frame_equal_mixed_dtypes(frame_or_series, any_numeric_ea_dtype, indexer
         tm.assert_equal(obj1, obj2, check_exact=True, check_dtype=False)
 
 
-def test_assert_series_equal_check_like_different_indexes():
+def test_assert_frame_equal_check_like_different_indexes():
     # GH#39739
     df1 = DataFrame(index=pd.Index([], dtype="object"))
     df2 = DataFrame(index=pd.RangeIndex(start=0, stop=0, step=1))
     with pytest.raises(AssertionError, match="DataFrame.index are different"):
         tm.assert_frame_equal(df1, df2, check_like=True)
+
+
+def test_assert_frame_equal_checking_allow_dups_flag():
+    # GH#45554
+    left = DataFrame([[1, 2], [3, 4]])
+    left.flags.allows_duplicate_labels = False
+
+    right = DataFrame([[1, 2], [3, 4]])
+    right.flags.allows_duplicate_labels = True
+    tm.assert_frame_equal(left, right, check_flags=False)
+
+    with pytest.raises(AssertionError, match="allows_duplicate_labels"):
+        tm.assert_frame_equal(left, right, check_flags=True)