Microsatellite SSR markers were developed and utilized to reveal the genetic diversity of 32 strains of Flammulina velutipes collected in Korea, China, and Japan. From SSR-enriched library, 490 white colonies were randomly selected and sequenced. In the 490 sequenced clones, 85 clones (17.35%) were redundant. Among the remaining 405 unique clones, 201 clones (49.6%) contained microsatellite sequences. As a result, 12 primer pairs produced reproducible polymorphic bands within diverse 4 strains and these selected markers were further characterized in 32 Flammulina velutipes strains. A total of 34 alleles were detected using the 12 markers, with an average of 3.42 alleles and the number of alleles ranged from two to seven per locus. The major allele frequency ranged from 0.42 (GB-FV-127) to 0.98 (GB-FV-166), and values for observed (HO) and expected (HE) heterozygosity ranged from 0.00 to 0.94 (mean = 0.18) and from 0.03 to 0.67 (mean = 0.32), respectively. SSR loci amplified with GB-FV-127 markers gave the highest polymorphism information content (PIC) of 0.61 and mean allele number of five, while for loci amplified with GB-FV-166 markers these values were the lowest, namely 0.03 and two. The mean PIC value (0.29) observed in the present study with average number of alleles (3.42). The genetic relationships among 32 Flammulina velutipes strains based on SSR data were generated by UPGMA cluster analysis. In conclusion, we succeeded in developing 12 polymorphic SSRs markers from SSR-enriched library of Flammulina velutipes. These SSRs are presently being used for phylogenic analysis and evaluation of genetic variations. In future, these SSR markers will be used in clarifying taxonomic relationships among the Flammulina velutipes.