We present phylogenetic analyses of the lizard genus Diplodactylus subgenus Strophurus using 1646 aligned positions of mitochondrial DNA sequences containing 893 parsimony-informative characters for samples of 12 species of Strophurus and 19 additional Australian gecko species. Sequences from three protein-coding genes (ND1, ND2 and COI) and eight intervening transfer RNA genes were examined using parsimony, maximum-likelihood and Bayesian analyses. Species of Strophurus appeared to form a monophyletic group with the possible exception of S. taenicauda. Strophurus has evolved two distinct defence/display characteristics: caudal glands, which expel an unpalatable substance, and striking mouth colours. Caudal glands appeared to have arisen once in a common ancestor of Strophurus, with dermal augmentation of caudal glands characterizing a subclade within the subgenus. Evolution of yellow and dark-blue mouth colours in Strophurus occurred in the context of diurnal activity and may be interpreted as an augmentation of defensive behavioural displays. Molecular divergence suggests that arboreality evolved in a common ancestor of Oedura and Strophurus approximately 29 Mya and that the caudal glands of Strophurus arose approximately 25 Mya. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82, 123–138.