We sequenced approximately 500 base pairs of DNA from the 16S region of the mitochondrial genome to estimate relationships among the freshwater crayfish genera of Australia and New Zealand. In total, 35 sequences were obtained, representing 32 species and all 10 genera native to Australia and New Zealand. From these sequences, maximum likelihood, minimum evolution and parsimony estimates of phylogenetic relationships among the genera were obtained and compared with previous hypotheses concerning the relationships among the crayfish genera. Our results support the monophyly of each genus (except perhaps Euastacus) and the organisation of these genera into three major clades: the first clade contains the genera Engaeus, Tenuibranchiurus, Geocharax, Gramastacus, and Cherax; the second clade contains the genera Paranephrops, Parastacoides, Euastacus, and Astacopsis; and the third clade contains the genus Engaewa. We reject the ecological hypothesis of Riek for two major clades of crayfish species. Finally, we provide a checklist of the Australian and New Zealand species as they are currently recognised.