We assessed genetic differentiation and habitat suitability for Homopus areolatus during current and Last Glacial Maximum (LGM) conditions. The ND4 locus retrieved two monophyletic mtDNA clades with lower diversity in clade 1, in the west, than in clade 2, in the south-east. Clade 1 showed a north-south and clade 2 a west-to-east genetic divergence, and the clades co-occur in the syntaxis zone of the Cape Fold Mountains. The clades occupy distinct niches with limited overlap. Rainfall seasonality contributed most to habitat suitability, with clade 1 being restricted to winter rainfall and clade 2 to all-year rainfall regions. Precipitation variables contributed 90% and 60%, respectively, to habitat suitability of clades 1 and 2, with temperature, particularly mean temperature of the driest quarter, being of greater importance for clade 2 than clade 1. Suitable habitat shrank from the LGM to current conditions, probably due to reduced rainfall in the west and higher temperatures in most regions. We conclude that patterns of genetic divergence are strongly associated with ecological niche divergence of H. areolatus clades. More studies are needed to assess the taxonomic status of clade 1, particularly in view of its shrinking habitat due to climate change and anthropogenic factors.