Japanese cultivated gentians (Gentiana triflora, G. scabra and their hybrids) are economically important floricultural plants and their use is growing annually worldwide. We isolated 48 simple sequence repeat (SSR) markers using a SSR-enriched genomic library constructed from a gentian double haploid (DH) line. We demonstrated the utility of these SSR markers as a molecular tool for identification of vegetative-propagated gentian cultivars that are difficult to distinguish from their morphology. Five SSR loci showed high genetic polymorphism among 12 Japanese gentian cultivars, ranging from five to eight alleles per locus, with expected heterozygosity between 0.656 and 0.826, and enabled simple and precise identification of cultivars. It was confirmed that the markers were suitable to estimate genetic similarity among cultivars and were also applicable to ten related Gentiana species. This is the first report of the development of a comprehensive set of SSR markers in Japanese cultivated gentians to facilitate genomic analyses in Gentiana, including species of economic importance.