diff --git a/spring-webmvc/src/main/java/org/springframework/web/servlet/resource/ResourceHttpRequestHandler.java b/spring-webmvc/src/main/java/org/springframework/web/servlet/resource/ResourceHttpRequestHandler.java
index ace74e2dfb..f0d420d79b 100644
--- a/spring-webmvc/src/main/java/org/springframework/web/servlet/resource/ResourceHttpRequestHandler.java
+++ b/spring-webmvc/src/main/java/org/springframework/web/servlet/resource/ResourceHttpRequestHandler.java
@@ -25,6 +25,7 @@ import java.util.HashMap;
 import java.util.List;
 import java.util.Locale;
 import java.util.Map;
+import java.util.stream.Collectors;
 
 import jakarta.servlet.ServletException;
 import jakarta.servlet.http.HttpServletRequest;
@@ -423,6 +424,7 @@ public class ResourceHttpRequestHandler extends WebContentGenerator
 	}
 
 	private void resolveResourceLocations() {
+		List<Resource> result = new ArrayList<>();
 		if (!this.locationValues.isEmpty()) {
 			ApplicationContext applicationContext = obtainApplicationContext();
 			for (String location : this.locationValues) {
@@ -451,7 +453,7 @@ public class ResourceHttpRequestHandler extends WebContentGenerator
 									"but resolved to a Resource of type: " + resource.getClass() + ". " +
 									"If this is intentional, please pass it as a pre-configured Resource via setLocations.");
 				}
-				this.locationsToUse.add(resource);
+				result.add(resource);
 				if (charset != null) {
 					if (!(resource instanceof UrlResource)) {
 						throw new IllegalArgumentException("Unexpected charset for non-UrlResource: " + resource);
@@ -460,7 +462,11 @@ public class ResourceHttpRequestHandler extends WebContentGenerator
 				}
 			}
 		}
-		this.locationsToUse.addAll(this.locationResources);
+
+		result.addAll(this.locationResources);
+		result = result.stream().filter(Resource::exists).collect(Collectors.toList());
+
+		this.locationsToUse.addAll(result);
 	}
 
 	/**
diff --git a/spring-webmvc/src/test/java/org/springframework/web/servlet/resource/ResourceHttpRequestHandlerTests.java b/spring-webmvc/src/test/java/org/springframework/web/servlet/resource/ResourceHttpRequestHandlerTests.java
index e7f87759cd..76c19d77a0 100644
--- a/spring-webmvc/src/test/java/org/springframework/web/servlet/resource/ResourceHttpRequestHandlerTests.java
+++ b/spring-webmvc/src/test/java/org/springframework/web/servlet/resource/ResourceHttpRequestHandlerTests.java
@@ -1,5 +1,5 @@
 /*
- * Copyright 2002-2020 the original author or authors.
+ * Copyright 2002-2021 the original author or authors.
  *
  * Licensed under the Apache License, Version 2.0 (the "License");
  * you may not use this file except in compliance with the License.
@@ -311,6 +311,24 @@ public class ResourceHttpRequestHandlerTests {
 		assertThat(this.response.getContentAsString()).isEqualTo("h1 { color:red; }");
 	}
 
+	@Test // gh-27538
+	public void filterNonExistingLocations() throws Exception {
+		List<Resource> inputLocations = Arrays.asList(
+				new ClassPathResource("test/", getClass()),
+				new ClassPathResource("testalternatepath/", getClass()),
+				new ClassPathResource("nosuchpath/", getClass()));
+
+		ResourceHttpRequestHandler handler = new ResourceHttpRequestHandler();
+		handler.setServletContext(new MockServletContext());
+		handler.setLocations(inputLocations);
+		handler.afterPropertiesSet();
+
+		List<Resource> actual = handler.getLocations();
+		assertThat(actual).hasSize(2);
+		assertThat(actual.get(0).getURL().toString()).endsWith("test/");
+		assertThat(actual.get(1).getURL().toString()).endsWith("testalternatepath/");
+	}
+
 	@Test
 	public void testInvalidPath() throws Exception {