Speciation rates vary greatly among taxa and regions and are shaped by both biotic and abiotic factors. However, the relative importance and interactions of these factors are not well understood. Here we investigate the potential drivers of speciation rates in South American freshwater fishes, the most diverse continental vertebrate fauna, by examining the roles of multiple biotic and abiotic factors. We integrate a dataset on species geographic distribution, phylogenetic, morphological, climatic, and habitat data. We find that Late Neogene-Quaternary speciation events are strongly associated with body-size evolution, particularly in lineages with small body sizes that inhabit higher elevations near the continental periphery. Conversely, the effects of temperature, area, and diversity-dependence, often thought to facilitate speciation, are negligible. By evaluating multiple factors simultaneously, we demonstrate that habitat characteristics associated with elevation, as well as body size evolution, correlate with rapid speciation in South American freshwater fishes. Our study emphasizes the importance of integrative approaches that consider the interplay of biotic and abiotic factors in generating macroecological patterns of species diversity.