In this short report, we documented the reappearance of the Eurasian beaver Castor fiber L. for the first time in Tuscany (Central Italy). After the detection of unequivocal signs of presence, we confirmed the occurrence of beavers through camera trapping. Genetic analyses (cytochrome-b mitochondrial gene) and hair microstructure confirmed it as the Eurasian species. The reports given here extend the current known range about 530 km south to the known extent of occurrence of this species. The presence of a relict population in this area is almost unlikely, despite being quite far from the nearest village; we therefore may suggest that the individuals occurring in Tuscany may be the result of local unauthorized releases. A precise number of free-ranging beavers remain unknown though at least two individuals are suspected to occur, based on body size. Apart from records of adult individuals, the presence of at least one juvenile suggests that reproduction in the wild may have occurred in 2020. Immediate actions should be promoted to monitor potential expansion, preserve this population, and/or to limit impacts on ecosystems and conflict with human activities.