The complex climatic and geological history of Southeast Asia has shaped this region’s high biodiversity. In particular, sea level fluctuations associated with repeated glacial cycles during the Pleistocene both facilitated, and limited, connectivity between populations. In this study, we used data from two mitochondrial and three anonymous nuclear markers to determine whether a fresh/brackish water killifish, Aplocheilus panchax, Hamilton, 1822, could be used to further understand how climatic oscillations and associated sea level fluctuations have shaped the distribution of biota within this region, and whether such patterns show evidence of isolation within palaeodrainage basins. Our analyses revealed three major mitochondrial clades within A. panchax. The basal divergence of A. panchax mitochondrial lineages was approximately 3.5 Ma, whilst the subsequent divergence timings of these clades occurred early Pleistocene (~2.6 Ma), proceeding through the Pleistocene. Continuous phylogeographic analysis showed a clear west-east dispersal followed by rapid radiation across Southeast Asia. Individuals from Krabi, just north of the Isthmus of Kra, were more closely related to the Indian lineages, providing further evidence for a freshwater faunal disjunction at the Isthmus of Kra biogeographic barrier. Our results suggest that Sulawesi, across the Wallace Line, was colonised relatively recently (~30 ka). Nuclear DNA is less geographically structured, although Mantel tests indicated that nuclear genetic distances were correlated with geographic proximity. Overall, these results imply that recent gene flow, as opposed to historical isolation, has been the key factor determining patterns of nuclear genetic variation in A. panchax, however, some evidence of historical isolation is retained within the mitochondrial genome. Our study further validates the existence of a major biogeographic boundary at the Kra Isthmus, and also demonstrates the use of widely distributed fresh/brackishwater species in phylogeographic studies, and their ability to disperse across major marine barriers in relatively recent time periods.