Phylogenetic relationships of the family Agamidae were inferred from 860 base positions of a mitochondrial DNA sequence of 12S and 16S rRNA genes. Results confirmed the monophyly of this family including Leiolepis and Uromastyx (Leiolepidinae), and indicated the sister relationship between Agamidae and Chamaeleonidae. Our results also indicated the presence of two major clades in Agamidae. In one of these major clades, “Leiolepidinae” was first diverged, followed by the Lophognathus and Hypsilurus in order, leaving Physignathus, Chlamydosaurus and Pogona as monophyletic. This result contradicts the currently prevailing hypothesis for the agamid phylogeny, which, on the basis of morphological data, assumes the primary dichotomy between Leiolepidinae and the remainder (Agaminae). The phylogenetic diversity of agamid lizards in the Australian region is supposed to have increased through an in situ continental radiation rather than through multiple colonizations from Southeast Asia. Distributions of some species in Asia and Melanesia are attributed to the secondary dispersals subsequent to this radiation.