We developed eight novel microsatellite markers for the salamander Hynobius tsuensis, which is endemic to Tsushima Island, Japan. In H. tsuensis, the number of alleles, observed heterozygosity, and expected heterozygosity in each locus ranged from 1 to 10, 0.00 to 0.76, and 0.00 to 0.76, respectively. In cross-species amplification, five and four of the eight loci were successfully amplified for all individuals of Hynobius sp. from Tsushima and H. nebulosus, respectively. Three species of Hynobius were clearly split from each other in the STRUCTURE analysis, indicating genetic differentiation in nuclear DNA among three species. This is the first report on genetic variation between two close relatives, Hynobius sp. from Tsushima and H. nebulosus based on nuclear loci. These novel markers will be useful for elucidating intraspecific variations and inferring conservation units within H. tsuensis, and studying intra- and inter-specific variations in other Hynobius.