Phylogenetic inferences based on molecular data provide support for the recognition of some Pangasius subgenera and, or species groups as distinct genera. It is suggested that Pangasiidae and Schilbeidae have diverged from a common ancestor probably since late Miocene. Moreover, the data provide evidence for the existence of two distinct episodes of speciation in Pangasiidae. The most ancient occurred in the middle Miocene and concerned emergence of the different genera, while the second referred to an intense speciation event in the genus Pangasius, probably between the early Miocene and the late Pliocene. Finally, this study revealed the existence of two new species of Pangasius.