The Lower Mekong River and its three major tributaries, the Sekong, Sesan, and Srepok rivers, are systems with high aquatic biodiversity that now face impacts from new hydroelectric dams. Despite the ecological, economic, and cultural importance of the freshwater biota, knowledge about aquatic ecology in this region remains poor. We used morphological and stable isotope data to explore how fish functional and trophic diversity vary between the four rivers that comprise the Mekong-3S river system. During our field surveys, the Sesan had experienced greatest flow alteration from dams and had lowest taxonomic and functional diversity, with species less packed and less evenly dispersed within morphological space compared to the other rivers. The Sekong had greatest functional diversity, with species less packed in morphological space. Species in the Mekong and Srepok were more evenly distributed in morphological space and had intermediate levels of functional diversity. Isotopic niche diversity in the Sesan did not appear to be significantly different from the Srepok and Sekong rivers. Conversely, the more species-rich Mekong fish assemblage encompassed a greater isotopic space with species less packed and evenly distributed. Greater trophic redundancy was observed amongst fishes of the 3S rivers than the Mekong. Species functional redundancy could buffer river food webs against species loss caused by dams; however, additional traits and niche dimensions should be evaluated to test this hypothesis. Overall, morphological and isotopic evidence from the Mekong and 3S rivers indicate that river impoundment and flow regulation function as an environmental filter that reduces fish functional diversity.