The Eurasian beaver (Castor fiber) represents an extraordinary example of demographic recovery after centuries of severe hunting and extirpation. Reintroduction programs and legal protection have triggered the recolonisation of Europe, with rapid expansion of beaver populations in the last 25 years. In northern Italy, the species reappeared in 2018 after a 450-years absence, due to natural spreading of individuals from neighbouring countries. Since 2021, the presence of beavers has been reported in several areas of central Italy, probably following unofficial releases. Here, we employed mitochondrial DNA markers, mainly from non-invasive samples, to rule out the presence of the North American beaver (C. canadensis) and to characterise both the beavers from northern Italy and the new population from central Italy, assessing their genetic relationships with other European beavers. The analysis of 35 samples from Italy and other countries provided no evidence of the North American sister taxon and revealed four widely distributed mitochondrial haplotypes, which are discussed in the current framework of genetically mixed populations in Europe. In addition, this note reports genetic data from beavers in the Balkans, where no information has been available so far.