Consistent, large-scale estimates of water availability are needed to identify and avoid potential conflicts among human and ecosystem uses of water. We present an assessment of water limitation, defined as the monthly balance (difference) between water supply (ws) and human consumptive water use (wc), for the conterminous United States (CONUS) during water years 2010–2020. We estimate that 26.7 million Americans, 8% of CONUS population, live in areas with chronic high or severe water limitation. Although ws greatly exceeds wc at the CONUS scale, water is limited locally or regionally due to spatial and temporal patterns in climate and wc. Our water limitation metric, the monthly supply and use index (SUI), peaked in 2012 during a widespread drought when 38% of the CONUS land area experienced elevated water stress. The central and Southwestern U.S. experienced the highest SUI due to the combination of low ws and high wc, especially for irrigation. Spatial overlays of SUI and fish habitat ranges, including those of conservation concern, revealed that several species had notable proportions of their habitat exposed to high or severe water limitation during spawning season over the modeled time period, especially the Arkansas River shiner. ws was calculated from two CONUS, physically-based, hydrologic models while wc was calculated from three CONUS models of water use for crop irrigation, thermoelectric power generation, and public supply. The ws and wc values were routed through a stream network and compared to calculate water limitation and SUI for human populations and fish species at the scale of 12-digit hydrologic unit codes. Evaluation of water availability at higher spatial and temporal resolution promotes more comprehensive analyses of the drivers of water availability and can be combined with complementary studies of water quality and water limiting thresholds to better understand the limitations on water availability.