© 2020 The Entomological Society of Japan Acilius kishii Nakane, 1963 (Coleoptera: Dytiscidae) is an endangered diving beetle species distributed in only one location, Lake Yashaga-Ike, Honshu Island, Japan. Acilius japonicus, which is related to A. kishii, is distributed widely in northern Honshu Island and Hokkaido Island in Japan. In this study, we identified 14 microsatellite loci for A. kishii and A. japonicus, including both polymorphic and monomorphic loci, using the next-generation sequencing method. We observed that 5 and 10 loci showed polymorphisms in 31 and 32 individuals of A. kishii and A. japonicus, respectively. The observed and expected heterozygosities were 0.00–1.00 and 0.00–0.74, respectively. These microsatellite loci could be useful for future conservation genetic studies, including monitoring of genetic diversity and extinction risk of A. kishii.