The white-clawed crayfish Austropotamobius pallipes species complex is widely distributed across the European area. This species has a narrow ecological niche and inhabits small streams with clean and cold waters. Due to anthropogenic activities, its population size and distribution have declined dramatically over the last fifty years. Moreover, pathogens carried by non-native invasive crayfish species caused the extinction of Austropotamobius populations in several areas of Italy. Residual isolated populations are now confined to high altitudes and pristine habitats, where the non-native crayfish have not yet reached. The Italian Peninsula has been identified as a repository of genetic diversity of Austropotamobius spp. So far, only A. pallipes has been included in the IUCN Red List of Threatened Species, which hinders conservation initiatives for other Italian species and subspecies that have not yet been formally recognised. The identification of management units is fundamental for conservation activities which can be achieved using genomic tools. To this end, a total of 1,650 samples of Austropotamobius spp. were collected from 75 populations distributed across Italy and Croatia and characterised with both Genotyping-By-Sequencing and COI mtDNA sequencing approaches. Here, we report the use of genome wide SNPs markers that clearly identified three distinct genetic clusters corresponding to A. pallipes, Austropotamobius fulcisianus fulcisianus and A. fulcisianus meridionalis. This provides genome wide evidence of the presence of two species (A. pallipes and A. fulcisianus fulcisianus) and suggests that A. fulcisianus meridionalis may warrant recognition as a distinct species. These findings, are corroborated by COI mtDNA results. We also show that there is extensive genetic differentiation between local population groups that roughly correspond to different water basins, which lays the foundations for targeted reintroductions of Austropotamobius spp. populations guided by the distribution of genomic diversity.