Nuclear DNA (nDNA) markers were developed to distinguish between the closely related brackish water gobies Tridentiger brevispinis and T. obscurus. Although genetic differentiation of the two species has already been demonstrated by allozyme analysis in previous studies, the nucleotide sequences of mitochondrial DNA (mtDNA) haplotypes were similar and often shared by introgressive hybridization, obscuring the identification of the two species by mtDNA markers. In this study, one mtDNA gene [cytochrome b (cytb)] and four nuclear DNA gene regions [G protein-coupled receptor 85 (gpr85), ryanodine receptor 3 (ryr3), recombination activating protein 1 (rag1) and zic family member 1 (zic1)] were sequenced in 11 to 17 individuals, respectively, of T. brevispinis and T. obscurus, collected from the Mukogawa River, Hyogo Prefecture, Japan. The results for the mtDNA cytb region matched those of previous studies, the nucleotide sequences being very similar, with haplotypes shared among species. On the other hand, two (gpr85 and ryr3) of the four nDNA regions clearly differed between the two species, PCR-RFLP conducted on the former also showing specifically-different electrophoretic patterns. In order to confirm that nDNA PCR-RFLP could distinguish between the two species in other populations, additional samples of both from the Shonai River, Aichi Prefecture were subjected to and identified by the above method. In addition, eight individuals of putative F1 hybrid identified by allozyme analysis (three diagnostic loci) were also investigated. Although six of the eight putative hybrids included heterozygotes at both of the two nDNA PCR-RFLP loci, two individuals were characterized by a heterozygotic pattern at one locus, homozygotic at the other, both individuals possibly being F2 or backcross progeny, although initially misidentified as F1. The results indicated that nDNA markers may be helpful in distinguishing closely related Tridentiger species, which cannot be identified by mtDNA markers.