Although landscape features such as mountains and rivers are recognized often as limiting factors to amphibian dispersal and gene lfow, a limited number of studies have investigated such patterns across Southeast Asia. A perfect example of this is Thailand, located in one of the world’s biodiversity hotspot regions. Thailand represents the corridor between mainland Asia and the Sunda Shelf, a famous and widely recognized biogeographic region, and yet there are few studies on the genetic structure among populations of amphibian species distributed across Thailand. The Southeast Asian tree frog, Chiromantis hansenae has been reported to possess a geographic range that is restricted to Thailand and, presumably, Cambodia. Here, we investigate phylogenetic relationships among C. hansenae populations using partial sequences of the mitochondrial 16S rRNA gene and nuclear POMC gene. Our results reveal two distinct evolutionary lineages within C. hansenae populations in Thailand. The genetic divergence among populations between these two clades is considerable, and results support inter-population divergence, and high genetic differentiation (pairwise FST=0.97), between two localities sampled in western Thailand (TK1 and TK2), separated from each other by 40 kilometers only. The results suggest that landscape features across Thailand may have a profound impact on patterns of diversiifcation in the country, underscoring the urgent need for ifne-scale investigations of genetic structure of endemic and“widespread”species.