Sequence data derived from a 306 bp fragment of mitochondrial cytochrome b and molecular variance estimates were used to investigate the genetic population structure of the endangered and endemic lizard Podarcis atrata of the Columbretes archipelago (Mediterranean, Spain). Our results show a very high and significant among-population genetic differentiation. FST values and phylogenetic analyses confirm the evolutionary distinctiveness of P. atrata populations, suggesting that the populations of these islands deserve special protection measures. The populations of the two islands Columbrete Grande and Mancolibre are less differentiated than those of Foradada and Lobo, and seem to have retained mainland haplotypes. This situation needs further attention as the origin of the mainland haplotypes is still unclear. If they are a result of recent introductions from mainland specimens, then they may represent a threat to the endemic lizards of the Columbretes islands.