Previous genetic investigations of the Mekong giant catfish ( Pangasianodon gigas Chevey, 1913) and striped catfish ( Pangasianodon hypophthalmus Sauvage, 1878) provided discordant results. Here, we sequenced mitochondrial (mt) DNA of the cytochrome B region, and a control region, to characterize the genetic variation of P. gigas, P. hypophthalmus, and hybrids of these two species. Among the three groups, P. hypophthalmus had the greatest diversity in both regions, yet all three studied groups showed lower genetic diversity compared to the results of previous studies. The Bayesian Skyline Plots showed a reduction in effective population sizes of the parental species. The hybrids were found to have a constant population size with a recent divergence time. As expected, the network and neighbor joining tree showed a close maternal genetic relationship between the hybrid and P. hypophthalmus, a reflection of the breeding between male P. gigas and female P. hypophthalmus. Our results provide genetic information on these endangered fish that will be useful for both conservation and commercial breeding programs.