We characterized eighteen water voles, Arvicola amphibius (s.l.), from five populations along the Italian peninsula by means of mtDNA cytochrome b (Cytb) sequences. The samples included aquatic voles and one fossorial population from northern Italy. The standard karyotype of four voles from one central Italian population was also analysed and was identical to the one found in other populations outside Italy. Phylogenetic analyses, including vole Cytb haplotypes from the entire range, indicated the existence of a well supported and highly divergent Italian lineage (4.3%), sister to all the other haplotypes. The fossorial voles are not genetically differentiated from the aquatic voles from a nearby population and belong to the same taxon. Given the high Cytb divergence and the results of previous investigations on allozymes and hybrid fertility, we believe that the Italian population of water voles belongs to a distinct species, Arvicola italicus Savi, 1838, with the type locality near Pisa, although a morphological assessment of the entire skull is necessary to define it.