The red-tongue viper(Gloydius ussuriensis) is one of only three species of the genus Gloydius found in South Korea. Gloydius ussuriensis has a narrow activity radius and is distributed nationwide, and this species was reported to have the largest population among the Korean species in genus Gloydius. Preliminary results of a phylogenetic analysis using part of the mitochondrial DNA indicated that domestic G. ussuriensis is not comprised of monophyletic groups, and morphological analysis showed differences between domestic populations. In this study, we developed 17 microsatellites for the analysis of G. ussuriensis genetic diversity based on these characteristics. These microsatellites were developed using six multiplex panels, which could be employed to validate 80 G. ussuriensis specimens from different geographical regions in South Korea. The average number of alleles per locus was 12.2 and ranged from 4 to 25 alleles; the observed heterozygosity ranged from 0.238 to 0.950 and the expected heterozygosity ranged from 0.213 to 0.933. As a result of assessing four inland populations, a high level of genetic diversity was confirmed. These newly developed markers will be useful for further studies on the population structure and evolutionary history of the G. ussuriensis.