Diplodactylid geckos offer a model system for investigating the biogeographic history of Australia and adaptive radiations in the arid zone, but there is considerable uncertainty in the systematics of several key genera. We used sequence data from mitochondrial DNA to carry out a comprehensive analysis of phylogenetic relationships of geckos in the genus Diplodactylus. Parsimony and Bayesian analyses were highly concordant and allocated all species to one of two monophyletic clades, one comprising the species placed in the vittatus and conspicillatus species groups, the other comprising species placed in the stenodactylus and steindachneri species groups, plus D. byrnei, formerly in the vittatus group. The distinctness of these two clades is supported by external morphology of the digits, body and limb proportions, and osteology of the bones in the orbital region, and we use these characters to formally define the two clades as genera. We revive and expand the genus Lucasium for D. byrnei, D. steindachneri and the stenodactylus group, with the other species staying in a redefined Diplodactylus. The monotypic Rhynchoedura is distinct from Lucasium, although the Bayesian mtDNA analysis (but not parsimony) gives some support for a sister-group relationship between Lucasium and Rhynchoedura. Molecular data suggest that each of these clades represents a distinct radiation into semiarid and arid terrestrial habitats during the mid-Tertiary, well before the hypothesised Pliocene onset of major aridification.