The amphidromous sleeper Eleotris oxycephala (Perciformes: Eleotridae) is mainly distributed along the Kuroshio Current in East Asia, and this current is thought to be the main driver of the species' dispersal. Due to anthropogenic environmental changes in rivers, E. oxycephala is ranked as a threatened or near-threatened species in the red lists of 12 prefectures in Japan. Moreover, there is concern that the species' dispersal pattern could be changed due to fluctuations in the Kuroshio Current caused by global warming. In this study, 40 microsatellite markers were developed for E. oxycephala, and their suitability was tested on 43 individuals from two populations of E. oxycephala from Kanagawa and Miyazaki Prefectures. The number of alleles, expected heterozygosity and fixation index at each locus were 2–10 (mean = 5.350), 0.034–0.860 (mean = 0.650) and -0.261–0.448 (mean = 0.065), respectively. Furthermore, there was a lack of genetic difference between the two populations (FST = 0.008, F'ST = 0.024), indicating widespread gene flow via the Kuroshio Current. These markers will be useful to evaluate the genetic structure and infer population demographic history of E. oxycephala populations, which may assist in the conservation of this species.