Potamonautid river crabs were collected from 9 localities in Mpumalanga and Northern Province, South Africa, in order to determine morphological and genetic variation among these populations. Based on the presence or absence of epibranchial teeth, gonopod 1 shape, and marked allozyme divergence, 2 distinct forms were identified. The electrophoretic analysis of 11 enzyme systems, representing 14 presumptive loci, indicated a genetic identity value (I) of 0.66 between the 2 forms and revealed the presence of 4 diagnostic loci. It was concluded that these forms represented populations of Potamonautes sidneyi and populations of a previously unknown species, which is described as Potamonautes unispinus, new species, in this paper. A key to the known potamonautid river crab species from Mpumalanga and Northern Province is also provided.