Korean rose bitterling (Rhodeus uyekii) is a freshwater fish endemic to Korea. Natural populations of this species have experienced severe declines as a result of habitat fragmentation and water pollution. To conserve and restore R. uyekii, the genetic diversity of this species needs to be assessed at the population level. Eighteen novel polymorphic microsatellite loci for R. uyekii were developed using an enriched partial genomic library. Polymorphisms at these loci were studied in 150 individuals collected from three populations.