Invasive species are one of the main causes of biodiversity loss, and rodents in particular are regarded as a real threat worldwide, especially to island ecosystems. The Tuscan Archipelago National Park is the largest in the Mediterranean basin, it harbours a large number of autochthonous endemic species, mostly reptiles and insects, and hosts many migratory birds during their seasonal movements. Although a number of sites in the Archipelago are under strict protection regimes, the invasive black rat Rattus rattus has significantly affected survival of local wildlife. As part of an eradication campaign conducted in 2012 and 2017, we assessed genetic diversity and population differentiation of black rats from a total of six locations on the largest Elba Island, a possible source of invasion, and the southern, small islands of Pianosa and Montecristo using six nuclear DNA microsatellite loci. We recorded a strong population structure and identified the islands of Elba, Pianosa and Montecristo as three distinct eradication units. Despite some degree of admixture was recorded on Elba, the largest island of the archipelago was unlikely the main source of invasive rats to Pianosa and Montecristo. We also recorded evidence of past reduction in population size, particularly in Montecristo, probably due to repeated past founding events. Biodiversity management plans should consider monitoring vessels arriving to the Tuscan Archipelago from the mainland and the major Tyrrhenian islands in order to limit alien invasion. Moreover, as reinvasion can occur a few years after eradication, regular monitoring should be conducted thus to rapidly intercept the arrival of new invaders.