The oily bittering Acheilognathus koreensis is a freshwater species that is endemic to Korea and is experiencing severe declines in natural populations as a result of habitat fragmentation and water pollution. For the conservation and restoration of this species, it is necessary to assess its genetic diversity at the population level. We developed 13 polymorphic microsatellite loci that were used to analyze the genetic diversity of two populations collected from the Kum River and the Tamjin River in Korea. All loci exhibited Mendelian inheritance patterns when examined in controlled crosses. Both populations revealed high levels of variability, with the number of alleles ranging from 3 to 20 and observed and expected heterozygosities ranging from 0.500 to 0.969 and from 0.529 to 0.938, respectively. None of the loci showed significant deviation from Hardy-Weinberg equilibrium, and one pair of loci showed significant linkage disequilibrium after Bonferroni correction. Pairwise $F_{ST}$ and genetic distance estimation showed significant differences between two populations. These results suggest that the microsatellites developed herein can be used to study the genetic diversity, population structure and conservation measure of A. koreensis.