Four species of portunid crabs from South Brazil were studied by allozyme electrophoresis. The morphological and genetic distinction of C. sapidus subspecies (C. s. sapidus and C. s. acutidens) was studied by multivariate analysis of 10 morphometric characters and by allozyme electrophoresis of eighteen enzymes. The genetic relationship among Arenaeus cribrarius, C. sapidus, C. danae, and C. ornatus was assessed by these enzymes in order to test previous hypotheses based on morphological similarity. The morphometric and genetic evidence showed that there is no reason to justify the subspecific status of C. c. sapidus and C. s. acutidens, showing that their slight morphological differences are determined by the environment. Arenaeus cribrarius showed low genetic similarity to Callinectes spp., which justified its generic status (I = 0.252–0.396). The most closely related species within Callinectes were C. danae and C. ornatus, as previously suggested by their general morphology, showing a genetic identity of 0.762. According to the “sloppy molecular clock,” their divergence happened before the elevation of the Isthmus of Panamá, at the end of the Pliocene, about 3.8 Myr, which is in accordance with the fossil record.