We present a new map depicting the first global biogeographic regionalization of Earth's freshwater systems. This map of freshwater ecoregions is based on the distributions and compositions of freshwater fish species and incorporates major ecological and evolutionary patterns. Covering virtually all freshwater habitats on Earth, this ecoregion map, together with associated species data, is a useful tool for underpinning global and regional conservation planning efforts (particularly to identify outstanding and imperiled freshwater systems); for serving as a logical framework for large-scale conservation strategies; and for providing a global-scale knowledge base for increasing freshwater biogeographic literacy. Preliminary data for fish species compiled by ecoregion reveal some previously unrecognized areas of high biodiversity, highlighting the benefit of looking at the world's freshwaters through a new framework.