diff --git a/litestar/contrib/opentelemetry/plugin.py b/litestar/contrib/opentelemetry/plugin.py
index 39c84afc46..6d5f34677c 100644
--- a/litestar/contrib/opentelemetry/plugin.py
+++ b/litestar/contrib/opentelemetry/plugin.py
@@ -42,7 +42,8 @@ def _pop_otel_middleware(middlewares: list[Middleware]) -> tuple[list[Middleware
         for middleware in middlewares:
             if (
                 isinstance(middleware, DefineMiddleware)
-                and middleware.middleware is OpenTelemetryInstrumentationMiddleware
+                and isinstance(middleware.middleware, type)
+                and issubclass(middleware.middleware, OpenTelemetryInstrumentationMiddleware)
             ):
                 otel_middleware = middleware
             else: