Populations of Potamonautes perlatus were sampled at various points along the Olifants Rivers (220 km), in the Western Cape to examine the degree of genetic variation. Allozyme electrophoresis revealed a near fixed allelic difference at one locus and extensive differentiation at another locus between populations from the upper and lower reaches of the Olifants River system. Limited hybridization in the region of population sympatry was observed. The narrow hybrid zone and the low occurrence of hybrids suggest selection against hybrids. These results provide evidence for two genetically distinct freshwater crab populations. We concluded that the genetic divergence between the two population groups represents a species boundary between the two populations.