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Islands are fascinating study systems for biogeography, allowing researchers to investigate patterns across organisms on a comparable geographical scale. They are also often important for conservation. Here, we present the first bio-inventory of the ant fauna of the Aeolian Islands, a Sicilian volcanic archipelago formed within the last million years. We documented a total of 40 species, including one first record for Italy (Lasius casevitzi). Mitochondrial DNA barcodes were obtained for all 40 taxa sampled on the islands, 13 of which were studied genetically for the first time. Mitochondrial DNA sequences of island specimens were compared with those of conspecific samples from other Aeolian Islands, Sicily and mainland Italy. Standardized photographical documentation of all sequenced specimens is provided. All but one currently recognized species (97.5%) were recovered as monophyletic. Genetic divergence within species ranged up to 12.4% in Pheidole pallidula, although most species had much lower levels of intraspecific divergence. At the scale of the Aeolian Islands, intraspecific genetic divergence varied significantly between subfamilies, with species of the subfamily Myrmicinae showing higher intraspecific divergences than the Formicinae. Comparison of specimens from the Aeolian Islands with conspecific ones from the putative source populations (Sicily and mainland Italy) suggested that the island of Panarea has the genetically most derived myrmeco-fauna among the seven focal Islands. Overall, DNA barcoding is a useful-albeit not perfect-method for classifying poorly studied groups of organisms and ants in particular.