Although habitat fragmentation fosters extinctions, it also increases the probability of speciation by promoting and maintaining divergence among isolated populations. Here we test for the effects of two isolation factors that may reduce population dispersal within river networks as potential drivers of freshwater fish speciation: 1) the position of subdrainages along the longitudinal river gradient, and 2) the level of fragmentation within subdrainages caused by natural waterfalls. The occurrence of native freshwater fish species from 26 subdrainages of the Orinoco drainage basin (South America) was used to identify those species that presumably arose from in-situ cladogenetic speciation (i.e. neo-endemic species; two or more endemic species from the same genus) within each subdrainage. We related subdrainages fish diversity (i.e. total, endemic and neo-endemic species richness) and an index of speciation to our two isolation factors while controlling for subdrainages size and energy availability. The