Podarcis filfolensis is an endemic lizard from the Maltese archipelago. There is evidence of human-mediated decline and even extirpation of some insular populations of this species. However, information about the intraspecific genetic diversity and phylogeographic patterns of this species is limited. Here we analyze genetic markers from a multi-locus dataset (mtDNA, 2,533 bp; nuclear c-mos gene, 353 bp; 11 microsatellites) for individuals from extant populations of P. filfolensis. Despite generally low genetic variability, two main mitochondrial groupings were clearly identified. In general, individuals from the main island of Malta were genetically distinct from those from Gozo, Comino, Cominotto and Small Blue Lagoon Rock, and also from Linosa and Lampione individuals. Three genetic clusters were detected based on microsatellite data: one was found at higher frequency on Malta, while the other two included samples from the remaining islands, showing some concordance with the mtDNA pattern. A time-calibrated Bayesian tree for the principal mitochondrial lineages indicated strong statistical support for two P. filfolensis lineages that originated in the Pleistocene (105.4–869 Ka). We show that these lineages largely meet the criteria for recognition as evolutionary significant units despite some recent admixture (possibly due to recent translocations between islands). Human disturbance, low genetic variability, evidence of bottlenecks and extirpation on one island indicate that a thorough review of the current conservation status of P. filfolensis would be timely.