diff --git a/Lib/tempfile.py b/Lib/tempfile.py
index e6fb3c8e9a..2c7d546693 100644
--- a/Lib/tempfile.py
+++ b/Lib/tempfile.py
@@ -81,6 +81,12 @@ else:
         fd = _os.open(fn, _os.O_RDONLY)
         _os.close(fd)
 
+if _os.altsep is None:
+    _path_separators = (_os.sep,)
+else:
+    _path_separators = (_os.sep, _os.altsep)
+
+
 def _exists(fn):
     try:
         _stat(fn)
@@ -121,6 +127,12 @@ def _sanitize_params(prefix, suffix, dir):
             prefix = template
         else:
             prefix = _os.fsencode(template)
+    if output_type is str:
+        if any(sep in prefix for sep in _path_separators):
+            raise ValueError("Prefix contains system separator character")
+    else:
+        if any(_os.fsencode(sep) in prefix for sep in _path_separators):
+            raise ValueError("Prefix contains system separator character")
     if dir is None:
         if output_type is str:
             dir = gettempdir()
diff --git a/Lib/test/test_tempfile.py b/Lib/test/test_tempfile.py
index e5098d2eea..6d2ab60692 100644
--- a/Lib/test/test_tempfile.py
+++ b/Lib/test/test_tempfile.py
@@ -561,6 +561,23 @@ class TestGetTempPrefix(BaseTestCase):
             os.rmdir(d)
 
 
+class TestSanitizeParamsInner(BaseTestCase):
+    """Test the internal function _sanitize_params."""
+
+    def test_throw_exception_on_path_separator_detection(self):
+        with self.assertRaises(ValueError):
+            tempfile.mkstemp(prefix=f"{os.sep}home")
+
+    def test_throw_exception_on_encoded_path_separator_detection(self):
+        with self.assertRaises(ValueError):
+            tempfile.mkstemp(prefix=f"{os.fsencode(os.sep)}home")
+
+    @unittest.skipIf(os.altsep is None, "os.altsep is not present on this platform")
+    def test_throw_exception_on_alternative_path_separator_detection(self):
+        with self.assertRaises(ValueError):
+            tempfile.mkstemp(prefix=f"{os.altsep}home")
+
+
 class TestGetTempDir(BaseTestCase):
     """Test gettempdir()."""