Mitochondrial DNAs (mtDNAs) from nine morphologically distinct unisexual species and five bisexual species of lizards, all from the sexlineatus species-group of Cnemidophorus, were compared using restriction endonucleases. The unisexual lizards have mtDNAs that are identical at all or nearly all of the 128 sites cleaved. Although differing little in sequence, some mtDNAs differed in size due to the presence of tandem sequence duplications. Phylogenetic analysis of cleavage maps indicates that the mtDNAs of the unisexuals are most similar to that of the bisexual species C. inornatus. Considerable mtDNA diversity exists among C. inornatus populations, and one geographically restricted subspecies, C. i. arizonae, was identified as the most probable maternal ancestor of all nine unisexuals. All but one of these are triploid, and all have at least one C. inornatus gene complement. This, together with the homogeneity of their mtDNAs, suggests that all stem from one or a small number of allodiploid females (presumably parthenogenetic) that originated in a restricted geographic area in the recent past. These data, when combined with those from allozyme studies, preclude the possibility that most of the triploid unisexuals could have arisen via fertilization of an unreduced diploid ovum from one species by a haploid sperm from a different species.