Characterizing the genetic outfit of a species is fundamental to evaluate its status and devise optimal conservation plans to maintain or enhance the viability of its populations. The greater horseshoe bat (Rhinolophus ferrumequinum) underwent declines across Europe over the 19th century and was thus listed in the EU Habitat Directive and the species protection legislation of EU countries. To understand what is the state of the species in Italy, we described the genetic variability and population structure derived from 327 bats sampled in twenty-two Italian colonies. The analysis of the first part of the mitochondrial DNA control region (525 bp) and a panel of 12 microsatellite loci revealed high genetic variability throughout the study area and the presence of two genetically distinct groups: the Sardinian and the peninsular pools. We could also identify indications of a genetic substructure within the peninsular population, with a significant divergence concerning colonies located at the northern borders. Notably, the relevant genetic differentiation between Italian and Sardinian colonies should be carefully taken into account for conservation planning.