Taxonomic uncertainty of the Asian tree frogPolypedates leucomystaxcomplex presents the challenging task of inferring its biogeographical history. Here, we describe its dispersion and the genetic relationships among different populations in Thailand, where we connect the population of theP. leucomystaxcomplex of the Sunda Islands to the Indochina (mainland) population based on analyses of 266 sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Our maternal genealogy implies that there are four well-supported lineages in Thailand, consisting of Northern A (clade A:Polypedatessp.), Nan (clade B:P.cf.impresus), Southern (clade C:P. cf.leucomystax) and Northern D (clade D:P.cf.megacephalus), with Bayesian posterior probability >0.9. Phylogeny and haplotype networks indicate that clades A, B and D are sympatric. In contrast, clade C (P. cf.leucomystax) and clade D (P.cf.megacephalus) are genetically divergent due to the geographical barrier of the Isthmus of Kra, resulting in an allopatric distribution. Climatic conditions, in particular differences in rainfall on each side of the Isthmus of Kra, may play an important role in limiting the immigration of both clades. For the within-populations of either clades C or D, there was no significant correlation between geographic and genetic distance by the isolation-by-distance test, indicating intraspecific-dispersal of each clade. Population expansion occurred in clade C, whereas clade D showed a constant population. Taken together, theP. leucomystaxcomplex in South East Asia may have diversified under climatic pressure, leading to allopatric and/or sympatric speciation.