diff --git a/spring-jdbc/src/main/java/org/springframework/jdbc/datasource/lookup/AbstractRoutingDataSource.java b/spring-jdbc/src/main/java/org/springframework/jdbc/datasource/lookup/AbstractRoutingDataSource.java
index cc71ba2c9d18..81aee2588bf6 100644
--- a/spring-jdbc/src/main/java/org/springframework/jdbc/datasource/lookup/AbstractRoutingDataSource.java
+++ b/spring-jdbc/src/main/java/org/springframework/jdbc/datasource/lookup/AbstractRoutingDataSource.java
@@ -116,6 +116,15 @@ public void setDataSourceLookup(@Nullable DataSourceLookup dataSourceLookup) {
 
 	@Override
 	public void afterPropertiesSet() {
+		initialize();
+	}
+
+	/**
+	 * Synchronizes targetDataSources to resolvedDataSources
+	 * and defaultTargetDataSource to resolvedDefaultDataSource.
+	 * @throws IllegalArgumentException in case of targetDataSources is null
+	 */
+	public void initialize() {
 		if (this.targetDataSources == null) {
 			throw new IllegalArgumentException("Property 'targetDataSources' is required");
 		}
diff --git a/spring-jdbc/src/test/java/org/springframework/jdbc/datasource/lookup/AbstractRoutingDataSourceTests.java b/spring-jdbc/src/test/java/org/springframework/jdbc/datasource/lookup/AbstractRoutingDataSourceTests.java
index 8dc23a3f9651..bdb8e5939314 100644
--- a/spring-jdbc/src/test/java/org/springframework/jdbc/datasource/lookup/AbstractRoutingDataSourceTests.java
+++ b/spring-jdbc/src/test/java/org/springframework/jdbc/datasource/lookup/AbstractRoutingDataSourceTests.java
@@ -146,6 +146,31 @@ protected Object determineCurrentLookupKey() {
 		assertThat(routingDataSource.determineTargetDataSource()).isSameAs(ds);
 	}
 
+	@Test
+	void testInitialize_synchronizeTargetDataSourcesToResolvedDataSources() {
+		AbstractRoutingDataSource routingDataSource = new AbstractRoutingDataSource() {
+			@Override
+			protected Object determineCurrentLookupKey() {
+				return null;
+			}
+		};
+
+		DataSource ds1 = new StubDataSource();
+		DataSource ds2 = new StubDataSource();
+
+		Map<Object, Object> targetDataSources = new HashMap<>();
+		targetDataSources.put("ds1", ds1);
+		targetDataSources.put("ds2", ds2);
+		routingDataSource.setTargetDataSources(targetDataSources);
+
+		routingDataSource.initialize();
+
+		Map<Object, DataSource> resolvedDataSources = routingDataSource.getResolvedDataSources();
+		assertThat(resolvedDataSources).hasSize(2);
+		assertThat(resolvedDataSources.get("ds1")).isSameAs(ds1);
+		assertThat(resolvedDataSources.get("ds2")).isSameAs(ds2);
+	}
+
 	@Test
 	public void notInitialized() {
 		AbstractRoutingDataSource routingDataSource = new AbstractRoutingDataSource() {
diff --git a/spring-r2dbc/src/main/java/org/springframework/r2dbc/connection/lookup/AbstractRoutingConnectionFactory.java b/spring-r2dbc/src/main/java/org/springframework/r2dbc/connection/lookup/AbstractRoutingConnectionFactory.java
index ef285a5c47f1..9d55d01edfaf 100644
--- a/spring-r2dbc/src/main/java/org/springframework/r2dbc/connection/lookup/AbstractRoutingConnectionFactory.java
+++ b/spring-r2dbc/src/main/java/org/springframework/r2dbc/connection/lookup/AbstractRoutingConnectionFactory.java
@@ -127,6 +127,14 @@ public void setConnectionFactoryLookup(ConnectionFactoryLookup connectionFactory
 
 	@Override
 	public void afterPropertiesSet() {
+		initialize();
+	}
+
+	/**
+	 * Synchronizes targetConnectionFactories to resolvedConnectionFactories
+	 * and defaultTargetConnectionFactory to resolvedDefaultConnectionFactory.
+	 */
+	public void initialize() {
 		Assert.notNull(this.targetConnectionFactories, "Property 'targetConnectionFactories' must not be null");
 
 		this.resolvedConnectionFactories = CollectionUtils.newHashMap(this.targetConnectionFactories.size());
diff --git a/spring-r2dbc/src/test/java/org/springframework/r2dbc/connection/lookup/AbstractRoutingConnectionFactoryUnitTests.java b/spring-r2dbc/src/test/java/org/springframework/r2dbc/connection/lookup/AbstractRoutingConnectionFactoryUnitTests.java
index ecadb882327b..0b7009b7fb58 100644
--- a/spring-r2dbc/src/test/java/org/springframework/r2dbc/connection/lookup/AbstractRoutingConnectionFactoryUnitTests.java
+++ b/spring-r2dbc/src/test/java/org/springframework/r2dbc/connection/lookup/AbstractRoutingConnectionFactoryUnitTests.java
@@ -182,6 +182,19 @@ public void shouldAllowModificationsAfterInitialization() {
 				.verifyComplete();
 	}
 
+	@Test
+	void testInitialize_shouldDetermineRoutedFactory() {
+		connectionFactory.setTargetConnectionFactories(
+				singletonMap("key", routedConnectionFactory));
+		connectionFactory.setConnectionFactoryLookup(new MapConnectionFactoryLookup());
+		connectionFactory.initialize();
+
+		connectionFactory.determineTargetConnectionFactory()
+				.contextWrite(Context.of(ROUTING_KEY, "key"))
+				.as(StepVerifier::create)
+				.expectNext(routedConnectionFactory)
+				.verifyComplete();
+	}
 
 	static class DummyRoutingConnectionFactory extends AbstractRoutingConnectionFactory {