The European rabbit Oryctolagus cuniculus comprises O. c. algirus, endemic to southwestern Iberia, and O. c. cuniculus, which inhabits northeastern Iberia and southwestern France, and it is considered as the source of all introduced populations worldwide. Rabbit populations have long been established for hunting purposes and/or subjected to supplementation with individuals from intensely marketed stocks, with Italy being not an exception. We genotyped 42 fecal samples and 23 specimens (1877-2022) resident in museum collections at the mitochondrial DNA Cytochrome-b gene to infer to which subspecies belong the rabbits from the islands of Gorgona, Capraia, Montecristo, Giglio, and Giannutri (Tuscan archipelago). The Wildlife Refuge Padule di Bolgheri was selected as nearby mainland counterpart, and its population (n = 10) investigated along with 45 GenBank sequences of individuals (also domestic) from different continents. All modern and ancient Tuscan specimens were assigned to O. c. cuniculus, an unexpected result for Capraia and Montecristo that were assumed to host O. c. algirus on the base of the available literature. The network and the Bayesian clustering defined three groups. Modern rabbits from northern Capraia and most of those from Gorgona, which hosted (1873-1986) or still host (since 1869) an agricultural penal colony, respectively, belonged to a line disclosed in all ancient specimens from Capraia and that was new for the subspecies. The remaining rabbits from Capraia and Gorgona and all those from Montecristo and Giglio were related to European conspecifics while those from Giannutri were close to all the domestic individuals, with Bolgheri representing a mix of these two groups. Overall, the restrictions due to the presence of the penitentiaries likely prevented Capraia and Gorgona from an extended genetic homogenization associated to restocking practices. More broadly, we provided further evidence that the human-mediated rabbit colonization across the Mediterranean was based on O. c. cuniculus only.