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Mitochondrial DNA analysis was used to investigate genetic variation in the bronze featherback fish Notopterus notopterus in Indochina. Five hundred and five fish samples collected from 11 localities were separated into three genetically differentiated intraspecific groups. Chao Phraya River samples formed a distinct group. Mekong River samples were clearly separated into two different groups, namely, middle and lower Mekong groups. The lower Mekong group was closely related to Malay Peninsula group, even though the two groups are separated by a tract of ocean. F-values (0.82-0.95) also confirmed genetic differentiation of the middle and lower Mekong groups. Although the two Mekong groups inhabit the same river basin, molecular clock calculations indicate they separated approximately 1.2 Mya, suggesting (1) evolutionary divergence before invasion of the Mekong River; (2) differences in migration routes to the river; and/or (3) differences in the timings of invasion. The complexities of historical events cannot be ignored in determinations of factors responsible for the exceptional biodiversity in the Mekong basin. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101, 575-582. [ABSTRACT FROM AUTHOR]