In order to understand the processes that generate and maintain diversity, it is important to disentangle the roles of ecology and geography in speciation. We investigated the biogeographical and ecological factors that influenced the diversification of tree frogs (genus Sarcohyla) in the Mexican highlands, a region with high levels of endemism. Using single nucleotide polymorphism data for 58 samples, we found support for seven distinct genetic clusters within the Sarcohyla bistincta species complex, corresponding to Sarcohyla calthula, Sarcohyla pentheter and five populations within S. bistincta. A species tree analysis using the multispecies coalescent model did not support the monophyly of the five S. bistincta populations. We used niche modelling to calculate the ecological overlap among lineages; we found a degree of overlapping for most of the lineages, suggesting that ecological differentiation did not play a key role in their genetic divergence. Speciation and population structure in the complex have been shaped primarily by geological events, landscape modifications and climate changes during the Pleistocene. We discuss the relevance of genetic diversity for inferring the degree of species threats and recovery for conservation assessments.