diff --git a/plugins/node/opentelemetry-instrumentation-express/src/instrumentation.ts b/plugins/node/opentelemetry-instrumentation-express/src/instrumentation.ts
index 3df15aa699..013650327c 100644
--- a/plugins/node/opentelemetry-instrumentation-express/src/instrumentation.ts
+++ b/plugins/node/opentelemetry-instrumentation-express/src/instrumentation.ts
@@ -29,6 +29,7 @@ import { AttributeNames } from './enums/AttributeNames';
 import {
   asErrorAndMessage,
   getLayerMetadata,
+  getLayerPath,
   isLayerIgnored,
   storeLayerPath,
 } from './utils';
@@ -125,7 +126,7 @@ export class ExpressInstrumentation extends InstrumentationBase<
         const layer = this.stack[this.stack.length - 1] as ExpressLayer;
         instrumentation._applyPatch(
           layer,
-          typeof args[0] === 'string' ? args[0] : undefined
+          getLayerPath(args)
         );
         return route;
       };
@@ -146,7 +147,7 @@ export class ExpressInstrumentation extends InstrumentationBase<
         const layer = this.stack[this.stack.length - 1] as ExpressLayer;
         instrumentation._applyPatch(
           layer,
-          typeof args[0] === 'string' ? args[0] : undefined
+          getLayerPath(args)
         );
         return route;
       };
@@ -168,7 +169,7 @@ export class ExpressInstrumentation extends InstrumentationBase<
         instrumentation._applyPatch.call(
           instrumentation,
           layer,
-          typeof args[0] === 'string' ? args[0] : undefined
+          getLayerPath(args)
         );
         return route;
       };
diff --git a/plugins/node/opentelemetry-instrumentation-express/src/utils.ts b/plugins/node/opentelemetry-instrumentation-express/src/utils.ts
index 7e51b84124..fb7446ba76 100644
--- a/plugins/node/opentelemetry-instrumentation-express/src/utils.ts
+++ b/plugins/node/opentelemetry-instrumentation-express/src/utils.ts
@@ -145,3 +145,24 @@ export const asErrorAndMessage = (
   error instanceof Error
     ? [error, error.message]
     : [String(error), String(error)];
+
+
+/**
+ * Extracts the layer path from the route arguments
+ *
+ * @param args - Arguments of the route
+ * @returns The layer path
+ */
+export const getLayerPath = (args: unknown[]) => {
+  if (typeof args[0] === 'string') {
+    return args[0];
+  }
+
+  if (Array.isArray(args[0])) {
+    return args[0].map((arg) => (
+      typeof arg === 'string' || arg instanceof RegExp ? arg : '')
+    ).join(',');
+  }
+
+  return;
+}
diff --git a/plugins/node/opentelemetry-instrumentation-express/test/express.test.ts b/plugins/node/opentelemetry-instrumentation-express/test/express.test.ts
index f573a4a669..241f35bec4 100644
--- a/plugins/node/opentelemetry-instrumentation-express/test/express.test.ts
+++ b/plugins/node/opentelemetry-instrumentation-express/test/express.test.ts
@@ -566,4 +566,39 @@ describe('ExpressInstrumentation', () => {
       },
     });
   });
+
+  it('should work with ESM usage and multiple routes including regex', async () => {
+    await testUtils.runTestFixture({
+      cwd: __dirname,
+      argv: ['fixtures/use-express-regex.mjs'],
+      env: {
+        NODE_OPTIONS:
+          '--experimental-loader=@opentelemetry/instrumentation/hook.mjs',
+        NODE_NO_WARNINGS: '1',
+      },
+      checkResult: (err, stdout, stderr) => {
+        assert.ifError(err);
+      },
+      checkCollector: (collector: testUtils.TestCollector) => {
+        const spans = collector.sortedSpans;
+
+        console.debug(spans);
+
+        assert.strictEqual(spans[0].name, 'GET /test/arr/:id,/\\\/test\\\/arr[0-9]*\\\/required(path)?(\\\/optionalPath)?\\\/(lastParam)?/');
+        assert.strictEqual(spans[0].kind, SpanKind.CLIENT);
+        assert.strictEqual(spans[1].name, 'middleware - query');
+        assert.strictEqual(spans[1].kind, SpanKind.SERVER);
+        assert.strictEqual(spans[1].parentSpanId, spans[0].spanId);
+        assert.strictEqual(spans[2].name, 'middleware - expressInit');
+        assert.strictEqual(spans[2].kind, SpanKind.SERVER);
+        assert.strictEqual(spans[2].parentSpanId, spans[0].spanId);
+        assert.strictEqual(spans[3].name, 'middleware - simpleMiddleware');
+        assert.strictEqual(spans[3].kind, SpanKind.SERVER);
+        assert.strictEqual(spans[3].parentSpanId, spans[0].spanId);
+        assert.strictEqual(spans[4].name, 'request handler - /test/arr/:id,/\\\/test\\\/arr[0-9]*\\\/required(path)?(\\\/optionalPath)?\\\/(lastParam)?/');
+        assert.strictEqual(spans[4].kind, SpanKind.SERVER);
+        assert.strictEqual(spans[4].parentSpanId, spans[0].spanId);
+      },
+    });
+  });
 });
diff --git a/plugins/node/opentelemetry-instrumentation-express/test/fixtures/use-express-regex.mjs b/plugins/node/opentelemetry-instrumentation-express/test/fixtures/use-express-regex.mjs
new file mode 100644
index 0000000000..e5aebae064
--- /dev/null
+++ b/plugins/node/opentelemetry-instrumentation-express/test/fixtures/use-express-regex.mjs
@@ -0,0 +1,67 @@
+/*
+ * Copyright The OpenTelemetry Authors
+ *
+ * Licensed under the Apache License, Version 2.0 (the "License");
+ * you may not use this file except in compliance with the License.
+ * You may obtain a copy of the License at
+ *
+ *      https://www.apache.org/licenses/LICENSE-2.0
+ *
+ * Unless required by applicable law or agreed to in writing, software
+ * distributed under the License is distributed on an "AS IS" BASIS,
+ * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
+ * See the License for the specific language governing permissions and
+ * limitations under the License.
+ */
+
+// Use express from an ES module:
+//    node --experimental-loader=@opentelemetry/instrumentation/hook.mjs use-express-regex.mjs
+
+import { promisify } from 'util';
+import { createTestNodeSdk } from '@opentelemetry/contrib-test-utils';
+
+import { HttpInstrumentation } from '@opentelemetry/instrumentation-http';
+import { ExpressInstrumentation } from '../../build/src/index.js';
+
+const sdk = createTestNodeSdk({
+  serviceName: 'use-express-regex',
+  instrumentations: [
+    new HttpInstrumentation(),
+    new ExpressInstrumentation()
+  ]
+})
+sdk.start();
+
+import express from 'express';
+import * as http from 'http';
+
+const app = express();
+
+app.use(async function simpleMiddleware(req, res, next) {
+  // Wait a short delay to ensure this "middleware - ..." span clearly starts
+  // before the "router - ..." span. The test rely on a start-time-based sort
+  // of the produced spans. If they start in the same millisecond, then tests
+  // can be flaky.
+  await promisify(setTimeout)(10);
+  next();
+});
+
+app.get(['/test/arr/:id', /\/test\/arr[0-9]*\/required(path)?(\/optionalPath)?\/(lastParam)?/], (_req, res) => {
+  res.send({ response: 'response' });
+});
+
+const server = http.createServer(app);
+await new Promise(resolve => server.listen(0, resolve));
+const port = server.address().port;
+
+await new Promise(resolve => {
+  http.get(`http://localhost:${port}/test/arr/requiredPath/optionalPath/lastParam`, (res) => {
+    res.resume();
+    res.on('end', () => {
+      resolve();
+    });
+  })
+});
+
+await new Promise(resolve => server.close(resolve));
+await sdk.shutdown();