diff --git a/doc/source/whatsnew/v1.3.0.rst b/doc/source/whatsnew/v1.3.0.rst
index 16f76651a65aa..3f12b1b21ff30 100644
--- a/doc/source/whatsnew/v1.3.0.rst
+++ b/doc/source/whatsnew/v1.3.0.rst
@@ -682,6 +682,7 @@ Other
 - Bug in :class:`.Styler` where copying from Jupyter dropped top left cell and misaligned headers (:issue:`12147`)
 - Bug in :meth:`DataFrame.equals`, :meth:`Series.equals`, :meth:`Index.equals` with object-dtype containing ``np.datetime64("NaT")`` or ``np.timedelta64("NaT")`` (:issue:`39650`)
 - Bug in :func:`pandas.util.show_versions` where console JSON output was not proper JSON (:issue:`39701`)
+- Bug in :method:`DataFrame.query`, where "python" ``parser`` would raise NotImplementedError for == and != (:issue:`40436`)
 - Bug in :meth:`DataFrame.convert_dtypes` incorrectly raised ValueError when called on an empty DataFrame (:issue:`40393`)
 
 
diff --git a/pandas/core/computation/expr.py b/pandas/core/computation/expr.py
index 02660539f4981..aee5c3442780a 100644
--- a/pandas/core/computation/expr.py
+++ b/pandas/core/computation/expr.py
@@ -384,8 +384,6 @@ class BaseExprVisitor(ast.NodeVisitor):
     unary_op_nodes_map = {k: v for k, v in zip(unary_ops, unary_op_nodes)}
 
     rewrite_map = {
-        ast.Eq: ast.In,
-        ast.NotEq: ast.NotIn,
         ast.In: ast.In,
         ast.NotIn: ast.NotIn,
     }
diff --git a/pandas/tests/frame/test_query_eval.py b/pandas/tests/frame/test_query_eval.py
index fdbf8a93ddddf..785ad1001f537 100644
--- a/pandas/tests/frame/test_query_eval.py
+++ b/pandas/tests/frame/test_query_eval.py
@@ -548,6 +548,13 @@ def test_query(self):
             df[df.a + df.b > df.b * df.c],
         )
 
+    def test_query_equality(self):
+        engine, parser = self.engine, self.parser
+        df = DataFrame({"x": np.random.choice(["A", "B", "C"], size=20)})
+        tm.assert_frame_equal(
+            df.query("x == 'A'", engine=engine, parser=parser), df[df.x == "A"]
+        )
+
     def test_query_index_with_name(self):
         engine, parser = self.engine, self.parser
         df = DataFrame(