Impacts of urban development on aquatic populations are often complex and difficult to ascertain, but population genetic analysis has allowed researchers to monitor and estimate gene flow in context of existing and future hydroelectric projects. The Lower Mekong Basin is undergoing rapid hydroelectric development with around 50 completed and under construction dams and 95 planned dams. We investigated the baseline genetic diversity of two exploited migratory fishes, the Mud Carp Henicorhynchus lobatus (five locations) and the Rat-faced Pangasiid Helicophagus leptorhynchus (two locations), in the Lower Mekong Basin using the genomic ddRAD method. In both species, fish sampled upstream of Khone Falls are differentiated from those collected at other sites, and Ne estimates at the site above the falls are lower than at other sites. This was the first study to utilize thousands of RAD-generated SNPs 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. Additionally, Hen. 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.