The Neotropics harbors a megadiverse ichthyofauna comprising over 6300 species with approximately 80% in just three taxonomic orders within the clade Characiphysi. This highly diverse group has evolved in tropical South America over tens to hundreds of millions of years influenced mostly by re‐arrangements of river drainages in lowland and upland systems. In this study, we investigate patterns of spatial diversification in Neotropical freshwater fishes in the family Curimatidae, a species‐rich clade of the order Characiformes. Specifically, we examined ancestral areas, dispersal events, and shifts in species richness using spatially explicit biogeographic and macroevolutionary models to determine whether lowlands–uplands serve as museums or cradles of diversification for curimatids. We used fossil information to estimate divergence times in BEAST, multiple time‐stratified models of geographic range evolution in BioGeoBEARS, and alternative models of geographic state‐dependent speciation and extinction in GeoHiSSE. Our results suggest that the most recent common ancestor of curimatids originated in the Late Cretaceous likely in lowland paleodrainages of northwestern South America. Dispersals from lowland to upland river basins of the Brazilian and Guiana shields occurred repeatedly across independently evolving lineages in the Cenozoic. Colonization of upland drainages was often coupled with increased rates of net diversification in species‐rich genera such as Cyphocharax and Steindachnerina. Our findings demonstrate that colonization of novel aquatic environments at higher elevations is associated with an increased rate of diversification, although this pattern is clade‐dependent and driven mostly by allopatric speciation. Curimatids reinforce an emerging perspective that Amazonian lowlands act as a museum by accumulating species along time, whereas the transitions to uplands stimulate higher net diversification rates and lineage diversification.

Data collection and analyses were performed by all authors of the paper.