Abstract Many studies on the diversity of the South American biota support the role of ecological and geological events as main drivers of species diversification. For many groups, geomorphological events are the key drivers of diversification, while the influence of Pleistocene climate oscillations is prominent for others. To precisely indicate which events were key for the development of the astonishing biodiversity in South America, studies on widely distributed species are paramount. One such species, the dwarf swamp frog Pseudopaludicola mystacalis (Leptodactylidae, Leiuperinae), is widely distributed in open habitats of South America and we herein investigate population differentiation and diversification in this species across its geographic range. We sequenced a 1374 bp mtDNA fragment from 64 specimens across 25 localities. We used population assignment and species delimitation methods to assess genetic structure and lineage limits across the species distribution. We estimated, for each lineage, intraspecific diversity, divergence times, and demographic histories. Our results recovered ten lineages with up to 5% of genetic divergence among them. Diversification occurred mainly during the Tertiary, suggesting that Miocene-Pliocene topographic events had a major influence on the diversification of P. mystacalis. Pleistocene climatic oscillations also played a role on evolutionary history of P. mystacalis, causing demographic changes in one lineage.