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The genetic structure and dispersal dynamics of reptile populations are profoundly influenced by natural processes and human activities. While natural dispersal is shaped by species’ characteristics and paleogeographical features, human-mediated translocations have become increasingly prevalent, posing ecological challenges. Mitochondrial genetic markers have been pivotal in untangling invasion pathways for various species. Our study focuses on the Dalmatian Algyroides, Algyroides nigropunctatus (Duméril & Bibron, 1839), a lizard species endemic to the Balkan Peninsula, where recent observations in the Apulian region of Italy suggest an introduced population. Genetic analyses employing two mtDNA markers (16S and ND4 genes) elucidate the phylogenetic relationships of the Dalmatian Algyroides and trace the geographic origin of the introduced population. Our findings reveal areas in western Greece and southwestern Albania as the most probable areas of the source population, while we identify two previously undetected geographical lineages in the native range, highlighting the complex evolutionary history of the species in the region. Additionally, indications of potential glacial refugia and post-glacial dispersal patterns shed more light on the species’ demographic dynamics.