The invention relates to an SSR-labeled fingerprint of shiitake mushroom L9319 strain, and an application thereof. The fingerprint is composed of 7 pairs of SSR-labeled specific allelic fragments developed according to a shiitake mushroom genomic sequence. The application comprises the steps that: SSR-labeled amplification is carried out upon a shiitake mushroom strain; and an obtained band type is compared with the fingerprint. If the band type is consistent with the fingerprint, the strain is a shiitake mushroom L9319 strain. Compared with a conventional morphological detection method, an antagonistic test, and mushroom fruiting trials, the application provided by the invention has the advantages of short detection time, high accuracy, and good repeatability. In 25 collected nationally validated shiitake mushroom main cultivated strains of our nation, the fingerprint has shiitake mushroom L9319 strain specificity.