The red-spotted apollo butterfly, Parnassius bremeri Bremer, 1864 (Lepidoptera: Papilionidae), has been listed as an endangered species in Korea. We developed microsatellite markers by nest-generation sequencing (NGS), selected 12 markers, and applied the markers to available South Korean populations to understand population genetic characteristics. The genotyping of 40 P. bremeri individuals from three localities in South Korea showed that at each locus, the observed number of alleles ranged from 17 to 43, the observed and expected heterozygosities were 0.84722-0.90556 and 0.76045-0.79208, respectively, and FIS was –0.155 to –0.121. STRUCTURE analysis supported the presence of two genetic pools in all three populations, although an immediate reason for this subdivision is not known. The population based FST, RST, and front wing length collectively suggest that at least Samchuk population in Gangwon Province has a significant distance. Further scrutinized analysis is undergoing.