The Italian peninsula represented one of the main glacial refugia during climatic oscillations of the Pleistocene, currently being a biodiversity hotspot. In this work, we analysed for the first time the genetic diversity of harvest mouse populations in Italy, and we compared them with those of the rest of Eurasia. Mitochondrial cytochrome-b gene was amplified from 12 samples from throughout the Italian range. We recorded a very low genetic diversity, in line with the rest of the harvest mouse range. In the comparative phylogenetic tree, Northern Italy samples clustered together as a sister group of the rest of Europe, whereas those from Central Italy clustered with Central Europe samples. Harvest mice have recently conquered Southern Europe, i.e., possibly at the start of the Holocene. The global genetic homogeneity might be due to accidental human-mediated introductions or to the sharp decline of the habitat of the harvest mouse, which may in turn have caused severe bottlenecks in the populations of this small rodent.