The genetic variability and presence of population substructuresin 4 native Northern Italian sheep breeds, Alpagota, Brogna,Foza, and Lamon, undergoing in situ conservation, and 1 widespreadItalian breed, Bergamasca, were studied by investigating 19microsatellite markers. The breeds showed considerable geneticvariability in terms of number of alleles and heterozygosity,with the exception of Alpagota, which was the least variable(0.607). Nevertheless, a significant deficit of heterozygoteswas observed in each breed due to rather increased levels ofinbreeding or to the presence of population substructures, probablycaused by increased genetic variation in the founder populations.The analyses evidenced clear genetic differentiation (FST =0.085), reduced levels of admixture, and presence of privatealleles among the breeds, confirming their genetic uniqueness.In particular, according to Reynolds genetic distances, Alpagotawas the most differentiated, perhaps because it had been bredmostly in a rather isolated area. Loss of any of the investigatedbreeds would result in a loss of genetic diversity ranging from0.5 to 1.6% of the total observed gene diversity. Results supportedthe decision to safeguard these breeds as important reservoirsof genetic diversity and suggested breeding and mating practicesto maintain variability and to overcome within-breed substructures.