Geckos of clade III of the Tarentola mauritanica species complex are widespread throughout southern Europe and northern Africa. We investigated the genetic variability of the Italian populations by performing a widespread sampling throughout the mainland and the two main islands of Sicily and Sardinia. We analysed 199 newly generated sequences of the mitochondrial 16S rRNA gene and 269 nuclear genotypes inferred from nine microsatellite loci from 307 individuals. We found 13 new mitochondrial haplotypes in Italy, whereas previous findings reported a single haplotype widespread throughout the country and in the rest of Europe, which currently make Italy the centre of genetic diversity of this taxon. There was no evidence of mitochondrial DNA structuring with geographic correlation. At the population genetic level, our multilocus approach based on nuclear markers returned a shallow genetic structure. Nonetheless, we disclosed the presence of at least four distinct genetic clusters (namely the Adriatic, two Tyrrhenian and the Calabrian clusters). Our findings do not support the two hypotheses proposed to explain the low level of mitochondrial polymorphism in this taxon, namely the genetic hitch‐hiking due to selective sweep and the historical human‐mediated colonization hypotheses. Based on the fossil record, the presence in Italy of this taxon since the Pleistocene Epoch is plausible. Given the discordance in genetic structure between mitochondrial and nuclear DNA, the exact role of the Italian Peninsula in shaping the observed patterns of genetic diversity during the Pleistocenic climatic oscillations needs further investigation.