The black shinnerPseudopungtungia nigraMori, 1935 is an endangered fish endemic to Korea. It lives in the narrow basin of the Geumgang River, Mangyeonggang River, and Ungcheoncheon Stream, which flow into the West Sea of Korea. One population ofP. nigrain Ungcheoncheon Stream has been locally exterminated once; it is now inhabiting the upper reaches of the dam through a restoration program. Efforts to identify and understand the genetic structure of these populations are important for conservation planning. Here, we analyzed genetic diversity using 21 microsatellite markers for 9 populations. The mean number of alleles ranged from 4.4 to 8.1, mean allelic richness ranged from 4.6 to 7.8, mean observed heterozygosity ranged from 0.519 to 0.702, and mean expected heterozygosity ranged from 0.540 to 0.763. All groups had recent and historical bottlenecks (P< 0.05, M-ratio < 0.68). Three groups [YD (2019), OC and UC] had significant inbreeding index values, suggesting that they were engaged in inbreeding. We observed a moderate level of genetic differentiation between MG and the rest of the population (FST= 0.135 to 0.168,P< 0.05). The genetic structure exhibited a fitting constantK= 2, along with separation between MG and the remaining populations. With respect to genetic flow, YD (2019), OC, CG, and ND shifted to the UC population (0.263 to 0.278). The genetic flow of each population was transferred only within the population; there was no gene flow among populations, except for the Ungcheoncheon Stream population. This study shows that the Ungcheoncheon Stream population needs conservation efforts to increase its genetic diversity, and the Geumgang River populations needs a conservation plan that considers the possibility of conservation and evolution through gene exchange among the populations.