We assessed how the variation in environmental factors influences mayfly assemblages in conserved streams of different Amazonian interfluves. We hypothesized that environmental variables, mayfly assemblages, and the influence of environmental factors vary among these interfluves, given their distinct evolutionary and ecological histories, and patterns of human interference. We also expected that the effects of environmental variation on species richness and composition would be greater among interfluves than among streams within the same interfluve. Our hypotheses were corroborated, reflecting the considerable heterogeneity of the Amazonian biome, where streams vary naturally among interfluves, determining broad scale divergences in species composition. The environment affected mayfly diversity in different ways among interfluves, reflecting the influence of historical processes that resulted in the formation of centers of endemism delimited by major Amazonian rivers, in which distinct biotic and abiotic factors have prevailed. These regional effects have resulted from the heterogeneity of environmental gradients, which affect mayfly diversity. The results of the present study indicate that, depending on the scale and topography of the interfluve, environmental predictors of the structure of mayfly assemblages vary considerably. This has important implications for the definition of control sites for the assessment and monitoring of environmental quality.