The family Aeglidae consists of a single genus (Aegla) of anomuran crabs restricted to the Neotropical region of South America. Much of the present-day distribution of freshwater species in southern Chile has been impacted by Pleistocene glacial cycles. The melting of ice sheets created elaborate lake and river systems throughout this area and played an important role in the speciation of Aegla. In this study, we sampled phylogenetically closely related species of Aegla from one river (A. cholchol) and one lake (A. rostrata), to examine the molecular divergence across three genetic loci (elongation factor 1 (EF1) intron, 16S, cytochrome c oxidase subunit I (COI)) and how genetic variation differs between the two habitats. We estimate the relative timing of divergence using Bayesian molecular dating methods and the associated molecular data. We then examine whether morphometrics allow differentiation between the two species and sexes.