diff --git a/alembic/config.py b/alembic/config.py
index 2c52e7cd..95502cb4 100644
--- a/alembic/config.py
+++ b/alembic/config.py
@@ -339,6 +339,27 @@ def messaging_opts(self) -> MessagingOptions:
             ),
         )
 
+    def get_separator_char(self, name: str) -> Optional[str]:
+        separator = self.get_main_option(name)
+
+        split_on_path = {
+            None: None,
+            "space": " ",
+            "newline": "\n",
+            "os": os.pathsep,
+            ":": ":",
+            ";": ";",
+        }
+
+        try:
+            return split_on_path[separator]
+        except KeyError as ke:
+            raise ValueError(
+                "'%s' is not a valid value for %s; "
+                "expected 'space', 'newline', 'os', ':', ';'"
+                % (separator, name)
+            ) from ke
+
 
 class MessagingOptions(TypedDict, total=False):
     quiet: bool
diff --git a/alembic/script/base.py b/alembic/script/base.py
index 30df6ddb..f556f354 100644
--- a/alembic/script/base.py
+++ b/alembic/script/base.py
@@ -180,51 +180,42 @@ def from_config(cls, config: Config) -> ScriptDirectory:
         version_locations_str = config.get_main_option("version_locations")
         version_locations: Optional[List[str]]
         if version_locations_str:
-            version_path_separator = config.get_main_option(
+            split_char = config.get_separator_char(
                 "version_path_separator"
             )
 
-            split_on_path = {
-                None: None,
-                "space": " ",
-                "newline": "\n",
-                "os": os.pathsep,
-                ":": ":",
-                ";": ";",
-            }
-
-            try:
-                split_char: Optional[str] = split_on_path[
-                    version_path_separator
-                ]
-            except KeyError as ke:
-                raise ValueError(
-                    "'%s' is not a valid value for "
-                    "version_path_separator; "
-                    "expected 'space', 'newline', 'os', ':', ';'"
-                    % version_path_separator
-                ) from ke
+            if split_char is None:
+                # legacy behaviour for backwards compatibility
+                version_locations = _split_on_space_comma.split(
+                    version_locations_str
+                )
             else:
-                if split_char is None:
-                    # legacy behaviour for backwards compatibility
-                    version_locations = _split_on_space_comma.split(
-                        version_locations_str
-                    )
-                else:
-                    version_locations = [
-                        x.strip()
-                        for x in version_locations_str.split(split_char)
-                        if x
-                    ]
+                version_locations = [
+                    x.strip()
+                    for x in version_locations_str.split(split_char)
+                    if x
+                ]
         else:
             version_locations = None
 
         prepend_sys_path = config.get_main_option("prepend_sys_path")
         if prepend_sys_path:
-            sys.path[:0] = list(
-                _split_on_space_comma_colon.split(prepend_sys_path)
+            split_char = config.get_separator_char(
+                "prepend_sys_path_separator"
             )
 
+            if split_char is None:
+                # legacy behaviour for backwards compatibility
+                sys.path[:0] = list(
+                    _split_on_space_comma_colon.split(prepend_sys_path)
+                )
+            else:
+                sys.path[:0] = (
+                    path.strip()
+                    for path in prepend_sys_path.split(split_char)
+                    if path
+                )
+
         rvl = config.get_main_option("recursive_version_locations") == "true"
         return ScriptDirectory(
             util.coerce_resource_to_filename(script_location),
diff --git a/tests/test_config.py b/tests/test_config.py
index a98994c4..e295f585 100644
--- a/tests/test_config.py
+++ b/tests/test_config.py
@@ -1,4 +1,5 @@
 import os
+import sys
 import tempfile
 
 from alembic import config
@@ -201,6 +202,104 @@ def test_version_locations(self, separator, string_value, expected_result):
             s = ScriptDirectory.from_config(cfg)
             eq_(s.version_locations, expected_result)
 
+    @testing.combinations(
+        (
+            "legacy raw string 1",
+            None,
+            "/foo",
+            ["/foo"],
+        ),
+        (
+            "legacy raw string 2",
+            None,
+            "/foo /bar",
+            ["/foo", "/bar"],
+        ),
+        (
+            "legacy raw string 3",
+            "space",
+            "/foo",
+            ["/foo"],
+        ),
+        (
+            "legacy raw string 4",
+            "space",
+            "/foo /bar",
+            ["/foo", "/bar"],
+        ),
+        (
+            "multiline string 1",
+            "newline",
+            " /foo  \n/bar  ",
+            ["/foo", "/bar"],
+        ),
+        (
+            "Linux pathsep 1",
+            ":",
+            "/Project A",
+            ["/Project A"],
+        ),
+        (
+            "Linux pathsep 2",
+            ":",
+            "/Project A:/Project B",
+            ["/Project A", "/Project B"],
+        ),
+        (
+            "Windows pathsep 1",
+            ";",
+            r"C:\Project A",
+            [r"C:\Project A"],
+        ),
+        (
+            "Windows pathsep 2",
+            ";",
+            r"C:\Project A;C:\Project B",
+            [r"C:\Project A", r"C:\Project B"],
+        ),
+        (
+            "os pathsep",
+            "os",
+            r"path_number_one%(sep)spath_number_two%(sep)s"
+            % {"sep": os.pathsep},
+            [r"path_number_one", r"path_number_two"],
+        ),
+        (
+            "invalid pathsep 2",
+            "|",
+            "/foo|/bar",
+            ValueError(
+                "'|' is not a valid value for prepend_sys_path_separator; "
+                "expected 'space', 'newline', 'os', ':', ';'"
+            ),
+        ),
+        id_="iaaa",
+        argnames="separator, string_value, expected_result",
+    )
+    def test_prepend_sys_path_locations(self, separator, string_value, expected_result):
+        cfg = config.Config()
+        if separator is not None:
+            cfg.set_main_option(
+                "prepend_sys_path_separator",
+                separator,
+            )
+        cfg.set_main_option("script_location", tempfile.gettempdir())
+        cfg.set_main_option("prepend_sys_path", string_value)
+
+        if isinstance(expected_result, ValueError):
+            message = str(expected_result)
+            with expect_raises_message(ValueError, message, text_exact=True):
+                ScriptDirectory.from_config(cfg)
+        else:
+            restore_path = list(sys.path)
+            try:
+                sys.path.clear()
+
+                ScriptDirectory.from_config(cfg)
+                eq_(sys.path, expected_result)
+            finally:
+                sys.path = restore_path
+
 
 class StdoutOutputEncodingTest(TestBase):
     def test_plain(self):