The complex climatic and geological history of Southeast Asia has been hypothesised to determine the most important aspects of the current phylogeographical structure and distribution of living organisms throughout the region. To test existing hypotheses, the genetic structure of the tire track eel, Mastacembelus favus, was investigated using 823 bp of mitochondrial DNA cytochrome b from 469 individuals from 51 localities encompassing its native range. The results classified all haplotypes into two major lineages, Lineage 1, which was further divided into Lineages 1a (lower Mekong, eastern Gulf of Thailand and Malay—Thai Peninsula), 1b (Ban- pakong River), 1c (Chao Phraya, Gulf of Thailand and Malay—Thai Peninsula) and 1d (Khlang Yai River), and Lineage 2, the upper reaches of the lower Mekong and the middle Mekong. Strong genetic discontinuities dated approximately 5 MYA were discovered in the Mekong with limited geographical overlap, suggesting a historically dissected drainage between two sections and species colonisation via different routes. The widespread Lineage 1 showed a strong signature of population expansion during the Pleistocene climate oscillation. Haplotype character- istics in the Malay—Thai Peninsula are hypothesised to result from postglacial dispersal from the Mekong and Chao Phraya through an extended Pleistocene drainage network.