Nematodes areabundant andubiquitous animals which are poorly known at intraspecific level. This work represents the first attempt to fill the gap on basic knowledge of genetic vari ability and differentiation in Protostrongylus oryctolagi, a nematode parasite of lagomorphs. 68 cox1sequences wereobtained from brownhares collected in five locations in Northern andCentral Italy, highlighting the presence of a high amount of genetic variation inside this species. The eleven haplotypes identified (Haplotype diversity equal to 0.702) were split into two lineages: lineage A (comprising six different haplotypes, A1-A6) and lineage B (B1-B5). Themeanintra-lineage amount of genetic variation was 0.3%, whereas the inter-lineage percentage of variation was ten-fold higher (3%). These two lineages were non-randomly distributed in the investigated areas. Lineage A showed a preference for Central Italy (Tus cany) even if it was sporadically found also in northern territories (Emilia-Romagna), while B-haplotypes were present exclusively in Emilia-Romagna. The analysis of molecular vari ance identified two main barriers to gene flow: (i) a strong major one which separate sam ples of Central Italy (PIA and GR7) from the northern ones (RE1, RE3 and MO1; Φ = 0.750, P = 0.00); (ii) a secondary faint barrier which separates Pianosa island from Grosseto (Φ ST =0.133, P=0.00). Any difference was found among northern samples (Φ ST ST =0.009, P =0.00). The observed data may beexplained by several factors ranging from the parasite’s biology (presence of a narrow host spectrum), the final host’s behaviour (small home range), the natural dispersion of the host-parasite dyad occurred in past or the recent pas sive men-mediated migration. Finally, the presence of unconventional shortened amplicons revealed the presence of NUMTs (nuclear copy of mitochondrial genes) in the P. oryctolagi nuclear genome, suggesting caution when using DNA barcode as unique marker for the identification of species belonging to this genus.