diff --git a/ophyd/areadetector/filestore_mixins.py b/ophyd/areadetector/filestore_mixins.py
index 9882ece78..602dbb201 100644
--- a/ophyd/areadetector/filestore_mixins.py
+++ b/ophyd/areadetector/filestore_mixins.py
@@ -225,6 +225,9 @@ def reg_root(self):
     def reg_root(self, val):
         if val is None:
             val = os.path.sep
+        root = PurePath(val)
+        if not root.is_absolute():
+            raise ValueError(f"The root part of the path must be absolute not {root=}.")
         self._root = PurePath(val)
 
     @property
diff --git a/ophyd/tests/test_areadetector.py b/ophyd/tests/test_areadetector.py
index 3903eaa68..5c1b949d2 100644
--- a/ophyd/tests/test_areadetector.py
+++ b/ophyd/tests/test_areadetector.py
@@ -768,6 +768,31 @@ def test_file_store_paths_can_change(mock_now: Mock) -> None:
         check_file_store_paths(file_store, test_case)
 
 
+def test_no_relative_root(
+    mock_now: Mock
+) -> None:
+    fs = DummyFS()
+
+    file_store = DummyFileStorePlugin(
+        name="test_file_store",
+        write_path_template="",
+        read_path_template="",
+        root=None,
+        reg=fs,
+    )
+    with pytest.raises(ValueError, match="The root part of the path must be absolute"):
+        file_store.reg_root = '.'
+
+    with pytest.raises(ValueError, match="The root part of the path must be absolute"):
+        file_store = DummyFileStorePlugin(
+            name="test_file_store",
+            write_path_template="",
+            read_path_template="",
+            root='.',
+            reg=fs,
+        )
+
+
 def check_file_store_paths(
     file_store: DummyFileStorePlugin,
     test_case: FileStorePathTestCase,