The iguanid subfamily Oplurinae consists of seven species of small to medium-sized, arboreal, sand-dwelling or rock-dwelling lizards endemic to Madagascar and the Comoros, belonging to the genera Oplurus and Chalarodon. We here present the first complete molecular species-level phylogeny for Oplurinae based on DNA sequences (865 bp) of the mitochondrial 16S rRNA gene and the nuclear c-mos gene. Our study is based on 52 specimens sampled from different populations in Madagascar and includes the geographically isolated population from Grande Comore, hitherto considered as subspecies Oplurus cuvieri comorensis or even as a separate species O. comorensis. Our results confirm that, within the genus Oplurus, the largely arboreal O. cuvieri and O. cyclurus form the sister clade to the remaining, more rock-dwelling species. Within the latter lineage, Oplurus quadrimaculatus is placed most basal, O. fierinensis and O. grandidieri are closely related sister species with high support, and O. saxicola is sister to the fierinensis/grandidieri lineage. Within the arboreal Oplurus, the Comoran sample shows no genetic differentiation relative to O. cuvieri populations from the North West and Sambirano regions of Madagascar, indicating that this population should not be considered as a separate species. In the monotypic genus Chalarodon, we discovered deep genetic divergences among populations of C. madagascariensis indicating the presence of a previously unrecognized cryptic species and the need for taxonomic revision.