diff --git a/scripts/pylib/twister/twisterlib/harness.py b/scripts/pylib/twister/twisterlib/harness.py
index 96b4a2e3461..6ae2622c810 100644
--- a/scripts/pylib/twister/twisterlib/harness.py
+++ b/scripts/pylib/twister/twisterlib/harness.py
@@ -245,19 +245,20 @@ class Pytest(Harness):
 
     def generate_command(self):
         config = self.instance.testsuite.harness_config
-        pytest_root = config.get('pytest_root', 'pytest') if config else 'pytest'
+        pytest_root = config.get('pytest_root', ['pytest']) if config else ['pytest']
         pytest_args = config.get('pytest_args', []) if config else []
         command = [
             'pytest',
             '--twister-harness',
             '-s', '-v',
-            os.path.join(self.source_dir, pytest_root),
             f'--build-dir={self.running_dir}',
             f'--junit-xml={self.report_file}',
             '--log-file-level=DEBUG',
             '--log-file-format=%(asctime)s.%(msecs)d:%(levelname)s:%(name)s: %(message)s',
             f'--log-file={self.pytest_log_file_path}'
         ]
+        command.extend([os.path.normpath(os.path.join(
+            self.source_dir, os.path.expanduser(os.path.expandvars(src)))) for src in pytest_root])
         command.extend(pytest_args)
 
         handler: Handler = self.instance.handler
diff --git a/scripts/schemas/twister/testsuite-schema.yaml b/scripts/schemas/twister/testsuite-schema.yaml
index 116c1a43379..96a121767a5 100644
--- a/scripts/schemas/twister/testsuite-schema.yaml
+++ b/scripts/schemas/twister/testsuite-schema.yaml
@@ -95,8 +95,10 @@ mapping:
             type: int
             required: false
           "pytest_root":
-            type: str
+            type: seq
             required: false
+            sequence:
+              - type: str
           "pytest_args":
             type: seq
             required: false
@@ -293,8 +295,10 @@ mapping:
                 type: int
                 required: false
               "pytest_root":
-                type: str
+                type: seq
                 required: false
+                sequence:
+                  - type: str
               "pytest_args":
                 type: seq
                 required: false
diff --git a/scripts/tests/twister/pytest_integration/test_harness_pytest.py b/scripts/tests/twister/pytest_integration/test_harness_pytest.py
index db7bf389fbd..e1b27a0cf02 100644
--- a/scripts/tests/twister/pytest_integration/test_harness_pytest.py
+++ b/scripts/tests/twister/pytest_integration/test_harness_pytest.py
@@ -48,6 +48,67 @@ def test_pytest_command(testinstance: TestInstance, device_type):
         assert c in command
 
 
+@pytest.mark.parametrize(
+    ('pytest_root', 'expected'),
+    [
+        (
+            ['pytest/test_shell_help.py'],
+            ['samples/hello/pytest/test_shell_help.py']
+        ),
+        (
+            ['pytest/test_shell_help.py', 'pytest/test_shell_version.py', 'test_dir'],
+            ['samples/hello/pytest/test_shell_help.py',
+             'samples/hello/pytest/test_shell_version.py',
+             'samples/hello/test_dir']
+        ),
+        (
+            ['../shell/pytest/test_shell.py'],
+            ['samples/shell/pytest/test_shell.py']
+        ),
+        (
+            ['/tmp/test_temp.py'],
+            ['/tmp/test_temp.py']
+        ),
+        (
+            ['~/tmp/test_temp.py'],
+            ['/home/joe/tmp/test_temp.py']
+        ),
+        (
+            ['$ZEPHYR_BASE/samples/subsys/testsuite/pytest/shell/pytest'],
+            ['/zephyr_base/samples/subsys/testsuite/pytest/shell/pytest']
+        ),
+        (
+            ['pytest/test_shell_help.py::test_A', 'pytest/test_shell_help.py::test_B'],
+            ['samples/hello/pytest/test_shell_help.py::test_A',
+             'samples/hello/pytest/test_shell_help.py::test_B']
+        ),
+        (
+            ['pytest/test_shell_help.py::test_A[param_a]'],
+            ['samples/hello/pytest/test_shell_help.py::test_A[param_a]']
+        )
+    ],
+    ids=[
+        'one_file',
+        'more_files',
+        'relative_path',
+        'absollute_path',
+        'user_dir',
+        'with_env_var',
+        'subtests',
+        'subtest_with_param'
+    ]
+)
+def test_pytest_handle_source_list(testinstance: TestInstance, monkeypatch, pytest_root, expected):
+    monkeypatch.setenv('ZEPHYR_BASE', '/zephyr_base')
+    monkeypatch.setenv('HOME', '/home/joe')
+    testinstance.testsuite.harness_config['pytest_root'] = pytest_root
+    pytest_harness = Pytest()
+    pytest_harness.configure(testinstance)
+    command = pytest_harness.generate_command()
+    for pytest_src in expected:
+        assert pytest_src in command
+
+
 def test_if_report_is_parsed(pytester, testinstance: TestInstance):
     test_file_content = textwrap.dedent("""
         def test_1():