We used ancient DNA techniques to amplify mitochondrial DNA fragments [cytochrome b (cyt b) and control region (CR)] from four museum specimens that represent, to our knowledge, the last testimony of the introduction of – now extinct – mongooses in Italy. We assessed the identity, origin and genetic variability of Italian mongooses using a species-level assignation Bayesian approach and haplotype network analysis. The genetic diversity (cyt b and CR) among Italian individuals was null. The cyt b sequences from the four museum specimens clearly established the Indian grey mongoose Herpestes edwardsii as the species having been introduced in Italy in the mid-XXth century. The latter had at least been released twice within a period of 10 years (Circeo NP and Capalbio, c. 200 km northward), probably from the same captive stock of the Giardino Zoologico di Roma. Assessment of genetic variability and haplotype network including native representatives of H. edwardsii suggested that the introduced pool likely originated from Pakistan or India. It appears that a combination of deleterious factors including low genetic diversity, restricted range and nonadaptation to western Palaearctic winter conditions is responsible for the extinction of the species in Italy. However, the lack of knowledge on the life-history traits and population dynamics of H. edwardsii prevents from further discussion of the factors that likely promoted its extinction process.