Background: Ecological specializations such as antipredator defense can reinforce morphological and distributional divergence within hybridizing species. Two hybridizing species of Daphnia (D. galeata and D. dentifera) are distributed in both Japan and North America; however, these populations have a longer history in Japan than in North America due to the differing impact of the last glaciation on these two regions. We tested the hypothesis that this longer coexistence in Japan would lead to extensive genetic admixture in nuclear and mitochondrial DNA whilst the distinct morphological traits and distributional patterns would be maintained. Results: The high level of correspondence among morphological traits, distribution, and mitochondrial and nuclear DNA types for the specimens with D. dentifera mtDNA indicated that the species distinction has been maintained. However, a discordance between mtDNA and nuclear ITS-1 types was observed for most specimens that had D. galeata mtDNA, consistent with the pattern seen between the two species in North America. This observation suggests nuclear introgression from D. dentifera into D. galeata without mitochondrial introgression. Conclusions: The separation of morphological traits and distribution ranges of the two hybridizing species in Japan, as well as in North America, has been maintained, despite large differences in climatic and geographical histories of these two regions. Variations in environmental factors, such as predation pressure, might affect maintenance of the distribution, although the further studies are needed to confirm this.