A molecular approach was employed to investigate stock structure in Siamese mud carp Henicorhynchus siamensis populations collected from 14 sites across mainland south-east Asia, with the major focus being the lower Mekong River basin. Spatial analysis of a mitochondrial DNA fragment (ATPase 6 and 8) identified four stocks in the Mekong River basin that were all significantly differentiated from a population in the nearby Khlong River, Thailand. In the Mekong River basin, populations in northern Lao People's Democratic Republic and northern Thailand represent two independent stocks, and samples from Thai tributaries group with those from adjacent Mekong sites above the Khone Falls to form a third stock. All sites below the Khone Falls constituted a single vast stock that includes Cambodia and the Mekong Delta in Vietnam. While H. siamensis is considered currently to undertake extensive annual migrations across the Mekong River basin, the data presented here suggest that natural gene flow may occur over much more restricted geographical scales within the basin, and hence populations may need to be managed at finer spatial scales than at the whole-of-drainage-basin level.