diff --git a/changelog/8014.trivial.rst b/changelog/8014.trivial.rst
new file mode 100644
index 000000000..3b9fb7bc2
--- /dev/null
+++ b/changelog/8014.trivial.rst
@@ -0,0 +1,2 @@
+`.pyc` files created by pytest's assertion rewriting now conform to the newer PEP-552 format on Python>=3.7.
+(These files are internal and only interpreted by pytest itself.)
diff --git a/src/_pytest/assertion/rewrite.py b/src/_pytest/assertion/rewrite.py
index 649726727..805d4c8b3 100644
--- a/src/_pytest/assertion/rewrite.py
+++ b/src/_pytest/assertion/rewrite.py
@@ -281,12 +281,16 @@ def _write_pyc_fp(
 ) -> None:
     # Technically, we don't have to have the same pyc format as
     # (C)Python, since these "pycs" should never be seen by builtin
-    # import. However, there's little reason deviate.
+    # import. However, there's little reason to deviate.
     fp.write(importlib.util.MAGIC_NUMBER)
+    # https://www.python.org/dev/peps/pep-0552/
+    if sys.version_info >= (3, 7):
+        flags = b"\x00\x00\x00\x00"
+        fp.write(flags)
     # as of now, bytecode header expects 32-bit numbers for size and mtime (#4903)
     mtime = int(source_stat.st_mtime) & 0xFFFFFFFF
     size = source_stat.st_size & 0xFFFFFFFF
-    # "<LL" stands for 2 unsigned longs, little-ending
+    # "<LL" stands for 2 unsigned longs, little-endian.
     fp.write(struct.pack("<LL", mtime, size))
     fp.write(marshal.dumps(co))
 
@@ -365,21 +369,33 @@ def _read_pyc(
     except OSError:
         return None
     with fp:
+        # https://www.python.org/dev/peps/pep-0552/
+        has_flags = sys.version_info >= (3, 7)
         try:
             stat_result = os.stat(os.fspath(source))
             mtime = int(stat_result.st_mtime)
             size = stat_result.st_size
-            data = fp.read(12)
+            data = fp.read(16 if has_flags else 12)
         except OSError as e:
             trace(f"_read_pyc({source}): OSError {e}")
             return None
         # Check for invalid or out of date pyc file.
-        if (
-            len(data) != 12
-            or data[:4] != importlib.util.MAGIC_NUMBER
-            or struct.unpack("<LL", data[4:]) != (mtime & 0xFFFFFFFF, size & 0xFFFFFFFF)
-        ):
-            trace("_read_pyc(%s): invalid or out of date pyc" % source)
+        if len(data) != (16 if has_flags else 12):
+            trace("_read_pyc(%s): invalid pyc (too short)" % source)
+            return None
+        if data[:4] != importlib.util.MAGIC_NUMBER:
+            trace("_read_pyc(%s): invalid pyc (bad magic number)" % source)
+            return None
+        if has_flags and data[4:8] != b"\x00\x00\x00\x00":
+            trace("_read_pyc(%s): invalid pyc (unsupported flags)" % source)
+            return None
+        mtime_data = data[8 if has_flags else 4 : 12 if has_flags else 8]
+        if int.from_bytes(mtime_data, "little") != mtime & 0xFFFFFFFF:
+            trace("_read_pyc(%s): out of date" % source)
+            return None
+        size_data = data[12 if has_flags else 8 : 16 if has_flags else 12]
+        if int.from_bytes(size_data, "little") != size & 0xFFFFFFFF:
+            trace("_read_pyc(%s): invalid pyc (incorrect size)" % source)
             return None
         try:
             co = marshal.load(fp)
diff --git a/testing/test_assertrewrite.py b/testing/test_assertrewrite.py
index 09383cafe..84d5276e7 100644
--- a/testing/test_assertrewrite.py
+++ b/testing/test_assertrewrite.py
@@ -2,6 +2,7 @@ import ast
 import errno
 import glob
 import importlib
+import marshal
 import os
 import py_compile
 import stat
@@ -1063,12 +1064,60 @@ class TestAssertionRewriteHookDetails:
         py_compile.compile(str(source), str(pyc))
 
         contents = pyc.read_bytes()
-        strip_bytes = 20  # header is around 8 bytes, strip a little more
+        strip_bytes = 20  # header is around 16 bytes, strip a little more
         assert len(contents) > strip_bytes
         pyc.write_bytes(contents[:strip_bytes])
 
         assert _read_pyc(source, pyc) is None  # no error
 
+    @pytest.mark.skipif(
+        sys.version_info < (3, 7), reason="Only the Python 3.7 format for simplicity"
+    )
+    def test_read_pyc_more_invalid(self, tmp_path: Path) -> None:
+        from _pytest.assertion.rewrite import _read_pyc
+
+        source = tmp_path / "source.py"
+        pyc = tmp_path / "source.pyc"
+
+        source_bytes = b"def test(): pass\n"
+        source.write_bytes(source_bytes)
+
+        magic = importlib.util.MAGIC_NUMBER
+
+        flags = b"\x00\x00\x00\x00"
+
+        mtime = b"\x58\x3c\xb0\x5f"
+        mtime_int = int.from_bytes(mtime, "little")
+        os.utime(source, (mtime_int, mtime_int))
+
+        size = len(source_bytes).to_bytes(4, "little")
+
+        code = marshal.dumps(compile(source_bytes, str(source), "exec"))
+
+        # Good header.
+        pyc.write_bytes(magic + flags + mtime + size + code)
+        assert _read_pyc(source, pyc, print) is not None
+
+        # Too short.
+        pyc.write_bytes(magic + flags + mtime)
+        assert _read_pyc(source, pyc, print) is None
+
+        # Bad magic.
+        pyc.write_bytes(b"\x12\x34\x56\x78" + flags + mtime + size + code)
+        assert _read_pyc(source, pyc, print) is None
+
+        # Unsupported flags.
+        pyc.write_bytes(magic + b"\x00\xff\x00\x00" + mtime + size + code)
+        assert _read_pyc(source, pyc, print) is None
+
+        # Bad mtime.
+        pyc.write_bytes(magic + flags + b"\x58\x3d\xb0\x5f" + size + code)
+        assert _read_pyc(source, pyc, print) is None
+
+        # Bad size.
+        pyc.write_bytes(magic + flags + mtime + b"\x99\x00\x00\x00" + code)
+        assert _read_pyc(source, pyc, print) is None
+
     def test_reload_is_same_and_reloads(self, pytester: Pytester) -> None:
         """Reloading a (collected) module after change picks up the change."""
         pytester.makeini(