ABSTRACT Dam development is fragmenting migratory pathways in the Mekong Basin, yet scientific understanding of Mekong fish migrations is limited. Therefore, the first‐ever transboundary acoustic telemetry network was deployed in the Mekong, Sekong, and Sesan rivers in Cambodia and Lao PDR to track movements of 81 individual fish representing 12 different species from June 2022 through March 2024. Upstream movement by Pangasius conchophilus during the transition between dry and wet seasons aligned with local ecological knowledge. Movements of Pangasius larnaudii between the Mekong and Sekong rivers represent a previously undocumented migratory pathway for this species. Long‐distance movements by Hemibagrus wyckioides into the 3S basin contradicted the assumption that the species moves only short distances. Together, these findings imply that fragmentation of riverine habitats from planned dams may lead to greater impacts on populations of these species than were formerly assumed.