Mitochondrial DNA sequences can be used to infer haplotype diversity in range-expanding species. The Eurasian beaver Castor fiber is experiencing a remarkable recovery in Europe following centuries of exploitation and local extinctions. The species has recently recolonized also Central Italy, likely due to unauthorized releases, whereas the first individuals occurring in North-Eastern Italy may be the results of natural range expansion from neighboring countries. Since the last months of 2023, presence signs were also observed in North-Western Italy, near the border with Switzerland. To assess the genetic diversity and origin of Italian beaver populations, we conducted mitochondrial DNA analysis on samples from this newly colonized area, revealing the occurrence of two mtDNA haplotypes belonging to the western mitochondrial lineage of the Eurasian beaver. Molecular sexing also confirmed the occurrence of both males and females. Therefore, monitoring this population should be urgently established following European laws. Future genetic investigations using nuclear DNA markers will be crucial for understanding the origin of these new populations and informing effective conservation strategies.