Polymerase chain reaction (PCR) restriction fragment length polymorphism analysis of mitochondrial DNA was applied to the genetic structure and evolutionary history of the more ancestral Pangasianodon hypophthalmus (n = 82), and the recently speciated catfish Pangasius bocourti (n = 90) from the Cambodian Mekong River. Both pangasiids were characterized by a lack of genetic population structure that may result from high levels of contemporary gene flow. Genetic diversity was lower in P. hypophthalmus than in P. bocourti. However, a different evolutionary history was inferred for both species based on genealogical and demographic analyses (mismatch analysis, Tajima's D- and Fu's F-S-tests). The genetic profile of the more ancestral P. hypophthalmus shows indications of a recent population bottleneck, whereas the recently speciated P. bocourti shows signatures of historical population expansion. This study stresses the importance of preserving the migration routes in the Cambodian Mekong basin in order to maintain the genetic diversity and long-term integrity of both species.