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In arid regions, spring-fed habitats are frequently the only year-round source of surface water and are essential habitats for aquatic organisms and primary water sources for terrestrial animals and human settlements. While these habitats have been relatively well-studied in some regions, those of the southern Sonoran Desert have received little attention. In 2008 and 2009, we documented the biodiversity of aquatic animals at 19 sites across three arid mountain ranges in Sonora, Mexico, characterized macrohabitat types, examined seasonal variation in aquatic invertebrate communities, and explored the effects of an exotic fish (tilapia) on native communities. We documented > 220 aquatic animal species, including several new species and range extensions for others. Macrohabitat type (oasis, tinaja, riffle, and seep) was more important than geographic location in structuring aquatic invertebrate communities at the scale of our study area (similar to 9,000 km(2)). We found little evidence of predictable seasonal variation in invertebrate communities, despite dramatic hurricane-induced flooding. Aquatic vertebrates were not diverse across the study region (4 amphibian species and 2 species each of fishes and reptiles), but were often locally abundant. Presence of non-native tilapia at one site was associated with reduced abundances of native leopard frogs and reduced richness and density of native aquatic invertebrates. The most pressing aquatic habitat conservation concerns in the region, as in other deserts, are groundwater withdrawal, unmanaged recreational visitation, and the introduction of exotic species. Spring-fed habitats around the world have been called hotspots of freshwater biodiversity, and those of the Sonoran Desert are no exception.