Genetic diversity was analysed in Salmo trutta populations living in an area of central Italy by sequencing 310 bp of the 5′ end of the mtDNA control region (D‐loop) and by Restriction Fragment Length Polymorphism (RFLP) analysis of three mtDNA segments. Data show a genetic structure profoundly altered by stockings with allochthonous material of Atlantic origin. In fact, 15 of the RFLP haplotypes detected are linked to an Atlantic sequence. The remaining 9 were instead coupled with sequences representing the three major phylogenetic lineages previously identified in the Mediterranean basin (Adriatic, marmoratus, and Mediterranean), representing the native genetic diversity of brown trout in that area. The close genetic affinity observed between these latter haplotypes and those found in the Balkan peninsula by other authors seems to be in accordance with a recent, natural, history of dispersion between the two borders of the Adriatic Sea. These results appear significant from a conservation point of view as, in spite of the massive stocking with hatchery‐reared specimens, they highlight the persistence of Salmo trutta native genetic diversity in central Italy.