We present a review of the scientific research needs for water sustainability of the Mekong River. One of the world’s longest rivers, millions depend daily on the Mekong for their food security and livelihoods, but the river is experiencing dramatic modifications, including extensive hydropower development. The 12 research challenges and themes presented here were identified by a diverse and interdisciplinary working group of 24 scientists with expertise in a broad range of scientific disciplines relevant to both physical and social dimensions of Mekong water sustainability, during a workshop held in Cambodia sponsored by the US National Science Foundation (NSF). The themes comprise a comprehensive research agenda that advocates an interdisciplinary, social-ecological approach. We describe the state of knowledge, and in doing so highlight key research needs and relevant literature. With many competing water needs, integrating these research needs into policy and management will be critical for future Mekong water sustainability.