Phylogenetic relationships among eight Brazilian genera of the lizard family Gymnophthalmidae were analyzed using mitochondrial DNA restriction-site data. The intergeneric relationships inferred from a cladistic analysis of 32 restriction sites were widely congruent with a recent phylogenetic analysis based on morphology. Assuming a molecular clock of 2% MY, these gymnophthalmid lizards may have diverged from a common ancestor some 7–8 MYA. The monophyly of the five eyelid-less microteiid genera from the Rio São Francisco Quaternary sand dunes was also supported by mitochondrial data, and the time estimated for this radiation was about 2–3 MYA. The mtDNA data also suggest that the unisexual population of Gymnophthalmus underwoodi from Roraima is not originated by hybridization between its two closely related sympatric bisexual species Gymnophthalmus leucomystax and Gymnopohthalmus vanzoi, in accordance with previous biochemical and karyotypic data.