diff --git a/src/_pytest/monkeypatch.py b/src/_pytest/monkeypatch.py
index <HASH>..<HASH> 100644
--- a/src/_pytest/monkeypatch.py
+++ b/src/_pytest/monkeypatch.py
@@ -36,7 +36,7 @@ def monkeypatch() -> Generator["MonkeyPatch", None, None]:
         monkeypatch.delattr(obj, name, raising=True)
         monkeypatch.setitem(mapping, name, value)
         monkeypatch.delitem(obj, name, raising=True)
-        monkeypatch.setenv(name, value, prepend=False)
+        monkeypatch.setenv(name, value, prepend=os.pathsep)
         monkeypatch.delenv(name, raising=True)
         monkeypatch.syspath_prepend(path)
         monkeypatch.chdir(path)
Docs: monkeypatch.setenv does not accept bool