South America contains three endemic genera of parastacid freshwater crayfishes (i.e., Parastacus Huxley, Samastacus Riek, and Virilastacus Hobbs), with a current total of ten described species. A previous author has argued that each of these genera has closer affinities with genera from Australia than to each other. We sequenced approximately 500 nucleotides of the 16S gene from mitochondrial DNA to estimate phylogenetic relationships among the South American genera of freshwater crayfishes and determined their phylogenetic positioning relative to the Australian genera. We sampled seven species representing all three genera from South America with 19 individuals. These sequences were combined with other Australasian crayfishes for a total representation of 54 sequences covering 13 genera. Our results indicate that the South American genera form a well-supported monophyletic group closely related to a subset of the Australasian crayfishes (Paranephrops and Parastacoides). Our results also provide strong support for the currently recognized generic designations.