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Current macroecological research places great emphasis on patterns of species richness (alpha diversity) and the underlying ecological and evolutionary processes involved in their origin and maintenance. However, few studies dealing with continental scales have addressed dissimilarities in species composition among areas (beta diversity). Using data for the occurrence of 3836 bird and 1641 mammal species in 4220 cells covering the New World, we assessed whether broad-scale macroecological patterns in beta diversity are related to dissimilarities in environmental variables and biotic units. We employed spatial regression and tree regression to model beta diversity. Difference in altitude was the best predictor of beta diversity. Accordingly, the highest beta diversity values were found in mountainous areas, particularly in the Andes, Central America and western North America. Explanatory variables related to transitions between biotic units (biome, ecoregion) were relatively unimportant. Areas that differ in altitude from their surroundings harbor different sets of species, and this may reflect either species adaptation to particular environmental conditions by range shifts, or species divergence by vicariance, or both.