Hydropower production is altering the Mekong River basin's riverine ecosystems, which contain the world's largest inland fishery and provide food security and livelihoods to millions of people. The basin's hydropower reservoir storage, which may rise from ~2% of its mean annual flow in 2008 to ~20% in 2025, is attenuating seasonal flow variability downstream of many dams with integral powerhouses and large storage reservoirs. In addition, tributary diversions for off-stream energy production are reducing downstream flows and augmenting them in recipient tributaries. To help manage tradeoffs between dam benefits (hydropower, irrigation, flood control, domestic water supply, and navigation) and their consequences for livelihoods and ecosystems, we review observed and projected impacts on river flows along both the Mekong mainstream and its tributaries. We include the effects of diversions and inter-basin transfers, which prior reviews of flow alteration in the Mekong basin have largely neglected. We also discuss the extent to which concurrent changes in climate, water demand, and land use, may offset or exacerbate hydropower-induced flow alteration. Our major recommendations for assessing hydrological impacts in the Mekong and other basins undergoing rapid hydropower development include synchronizing and integrating observational and modeling studies, improving the accuracy of reservoir water balances, evaluating multi-objective reservoir operating rules, examining hydropeaking-induced flow alteration, conducting multi-dam safety assessments, evaluating flow indicators relevant to local ecosystems and livelihoods, and considering alternative energy sources and reservoir sedimentation in long-term projections. Finally, we strongly recommend that dam impact studies consider hydrological alteration in conjunction with fish passage barriers, geomorphic changes and other contemporaneous stressors.