diff --git a/changelog_unreleased/yaml/16489.md b/changelog_unreleased/yaml/16489.md
new file mode 100644
index 000000000..53ab5c14c
--- /dev/null
+++ b/changelog_unreleased/yaml/16489.md
@@ -0,0 +1,16 @@
+#### Fix yaml comment in non-singleline items missing space before `#` (#16489 by @fyc09)
+
+<!-- prettier-ignore -->
+```yaml
+# Input
+123: # hello
+  # comment
+
+# Prettier stable
+123:# hello
+  # comment
+
+# Prettier main
+123: # hello
+  # comment
+```
diff --git a/src/language-yaml/print/mapping-item.js b/src/language-yaml/print/mapping-item.js
index 1a5be79ad..c9c4b408f 100644
--- a/src/language-yaml/print/mapping-item.js
+++ b/src/language-yaml/print/mapping-item.js
@@ -112,6 +112,8 @@ function printMappingItem(path, print, options) {
     implicitMappingValueParts.push(hardline);
   } else if (value.content) {
     implicitMappingValueParts.push(line);
+  } else if (hasTrailingComment(value)) {
+    implicitMappingValueParts.push(" ");
   }
   implicitMappingValueParts.push(printedValue);
   const implicitMappingValue = alignWithSpaces(
diff --git a/tests/format/yaml/mapping/__snapshots__/format.test.js.snap b/tests/format/yaml/mapping/__snapshots__/format.test.js.snap
index ffb14a8ab..aed27f30a 100644
--- a/tests/format/yaml/mapping/__snapshots__/format.test.js.snap
+++ b/tests/format/yaml/mapping/__snapshots__/format.test.js.snap
@@ -320,6 +320,39 @@ printWidth: 80
 ================================================================================
 `;
 
+exports[`comment-value-align.yml - {"tabWidth":4} format 1`] = `
+====================================options=====================================
+parsers: ["yaml"]
+printWidth: 80
+tabWidth: 4
+                                                                                | printWidth
+=====================================input======================================
+123: # hello
+  # comment
+
+=====================================output=====================================
+123: # hello
+    # comment
+
+================================================================================
+`;
+
+exports[`comment-value-align.yml format 1`] = `
+====================================options=====================================
+parsers: ["yaml"]
+printWidth: 80
+                                                                                | printWidth
+=====================================input======================================
+123: # hello
+  # comment
+
+=====================================output=====================================
+123: # hello
+  # comment
+
+================================================================================
+`;
+
 exports[`common.yml - {"tabWidth":4} format 1`] = `
 ====================================options=====================================
 parsers: ["yaml"]
diff --git a/tests/format/yaml/mapping/comment-value-align.yml b/tests/format/yaml/mapping/comment-value-align.yml
new file mode 100644
index 000000000..58cfb6489
--- /dev/null
+++ b/tests/format/yaml/mapping/comment-value-align.yml
@@ -0,0 +1,2 @@
+123: # hello
+  # comment