To examine how urban water use might affect freshwater biodiversity, we intersected our map of cities with insufficient water and a map of the freshwater ecoregions of the world. Freshwater ecoregions are spatial areas of similar ecology, often in the same major hydrologic drainage or of similar geology. If a city with insufficient water is located in a freshwater ecoregion, this means there is at least one stream in the ecoregion that is being fully used by urban residents. When an urban extent or its buffered area of water acquisition crossed the boundary of two freshwater ecoregions, in our calculation the population of the city with insufficient water was partitioned between the two ecoregions according to the volume of water available within that portion of each ecoregion. Population with insufficient water by freshwater ecoregion was graphically related to freshwater fish richness and endemism, because freshwater fish are one of the taxa most likely to be affected by water withdrawal.