Morphological and isozyme analyses were used to examine closely related species of Tridentiger obscurus and ulcorner: brevispinis, a pair of cryptic species which has intricate problem with regard to species boundary, and to clarify the taxonomic position. In this study, it was revealed from their 17 allopatric streams that there are differences of band ornamentation on the lateral side and three genetic markers between two species. However, genetic relationship between them (S = 0.813, D = 0.192) was within the intraspecific level of similarity. However, at the sympatry of two species no hybrid was found except at Bangjuk stream where the electrophoretic hybrids with slight deficiency of heterozygosity were observed. It is shown that at sympatry there is a finer microenvironmental subdivision between two species at all populations including Bangjuk stream. To manifest the extent of assortative mating, four syrnpatric subpopulations at Bangjuk stream were analyzed for departure from Hardy-Weinberg equillibrium by F-statistics. All subpopulations were deviated significantly from the expectation under random mating. The evidences presented here supports the recognition of Tridentiger obscurus and T. brevispinis as semispecies in the late stage of reproductive isolation