Southern Africa has a diverse endemic scorpion fauna, but a paucity of information currently confounds conservation of the group. Phylogeographic approaches represent a useful tool to identify the patterns and processes which underpin scorpion diversity, but these studies are lacking for southern African species. Among southern African scorpions, the semi-lithophilous Opistophthalmus pallipes has strict habitat requirements, and a distribution historically subjected to profound environmental turnover. As such, the species offers a model system to investigate the interplay between intrinsic and extrinsic factors as drivers of diversity and endemism. To investigate spatial genetic patterns within O. pallipes and the possible drivers thereof, the current study combines mitochondrial DNA and ecological information under a phylogeographic approach. The species is characterized by several genetically discrete and divergent populations. The factors which shape these genetic patterns appear to be both intrinsically (ecological specificity) and extrinsically (landscape structure and ecogeographic conditions) influenced, with major divergences corresponding to periods of profound environmental changes. Taken together, the findings of this study provide evidence of spatial genetic isolation and genetic diversity within a stenotopic southern African scorpion species. These findings partly explain the staggering diversity and endemism in southern African scorpions, but further phylogeographic studies are necessary to propose conservation scenarios for this group.