The authors investigated the baseline genetic diversity of two exploited migratory fishes, the mud carp Henicorhynchus lobatus (five locations), and the rat‐faced pangasiid catfish, Helicophagus leptorhynchus(two locations), in the Lower Mekong Basin using the genomic double digest restriction site‐associated DNA (ddRAD) sequencing method. In both species, fish sampled upstream of Khone Falls were differentiated from those collected at other sites, and N_e estimates at the site above the falls were lower than those at other sites. This was the first study to utilize thousands of RAD‐generated single nucleotide polymorphisms to indicate that the Mekong’s Khone Falls are a potential barrier to gene flow for these two moderately migratory species. The recent completion of the Don Sahong dam across one of the only channels for migratory fishes through Khone Falls may further exacerbate signatures of isolation and continue to disrupt the migration patterns of regionally vital food fishes. In addition, H. lobatus populations downstream of Khone Falls, including the 3S Basin and Tonle Sap system, displayed robust connectivity. Potential obstruction of migration pathways between these river systems resulting from future dam construction may limit dispersal, which has led to elevated inbreeding rates and even local extirpation in other fragmented riverine species.

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