The characterisation of intraspecific genetic diversity in representatives of the South American crayfish genus Parastacus Huxley, 1879 is here carried out for the first time by comparing populations of Parastacus brasiliensis (von Martens, 1869) as currently defined. Phylogenetic reconstructions based on mitochondrial and nuclear markers indicate the existence of multiple lineages, of which only one can be considered as P. brasiliensis sensu stricto. In addition, there are seven other lineages, one of which is the subspecies Parastacus brasiliensis promatensis Fontoura & Conter, 2008, which is here elevated to species level. We thereby increase to 14 the number of recognised species within Parastacus in South America. Genetic distances among P. brasiliensis sensu stricto and the lineages ‘A’, ‘B’ and ‘C’ increase with geographical distances, suggesting isolation by distance as an important driver of diversification, and eventually speciation, in these burrowing crayfishes. Parastacus brasiliensis occurs mainly in the Guaíba Lake basin and studied populations show limited connectivity and gene flow, probably due to habitat fragmentation. On the basis of genetic and distribution data, the conservation status of P. brasiliensis sensu stricto is confirmed as Near Threatened (NT). These findings lead us to encourage the establishment of preservation areas for isolated populations. The importance of preserving the newly recognised distinct gene pools in order to maintain overall genetic diversity is emphasised.