diff --git a/src/_pytest/main.py b/src/_pytest/main.py
index ad65ed29929..98e69ae28b5 100644
--- a/src/_pytest/main.py
+++ b/src/_pytest/main.py
@@ -386,6 +386,8 @@ def __init__(self, config):
 
         self.config.pluginmanager.register(self, name="session")
 
+        self._collected = set()
+
     def __repr__(self):
         return "<%s %s exitstatus=%r testsfailed=%d testscollected=%d>" % (
             self.__class__.__name__,
@@ -575,6 +577,10 @@ def _collect(self, arg):
             yield from m
 
     def _collectfile(self, path, handle_dupes=True):
+        key = (path, handle_dupes)
+        assert key not in self._collected, key
+        self._collected.add(key)
+
         assert (
             path.isfile()
         ), "{!r} is not a file (isdir={!r}, exists={!r}, islink={!r})".format(
diff --git a/testing/example_scripts/collect/collect_init_tests/conftest.py b/testing/example_scripts/collect/collect_init_tests/conftest.py
new file mode 100644
index 00000000000..365906d14a5
--- /dev/null
+++ b/testing/example_scripts/collect/collect_init_tests/conftest.py
@@ -0,0 +1,11 @@
+from typing import Set
+from typing import Tuple
+
+collected_already = set()  # type: Set[Tuple]
+
+
+def pytest_collect_file(path, parent):
+    # Ensure that the hook is only called once per path.
+    key = (path, parent)
+    assert key not in collected_already, key
+    collected_already.add(key)