Prions are responsible for transmissible spongiform encephalopathies (TSEs), also called scrapie in sheep. Single nucleotide polymorphisms (SNPs) in PRNP have been shown to play a crucial role in terms of incubation period and/or susceptibility to scrapie. Considering codons 136, 154, 171, five main alleles, associated with different degrees of susceptibility to classical scrapie have been detected. Homozygous ARR sheep are resistant to scrapie infection, however this genotype is not so frequent in some breeds. VRQ and ARQ alleles on the other hand are associated with a high susceptibility to scrapie. Additional polymorphisms within the ARQ allele, have been associated with scrapie resistance. Therefore, it is important to assess the frequency of both main and minor allelic variants, in order to develop a breeding programme based on genetic selection that simultaneously, increases scrapie resistance, and maintains a high variability of PRNP. Allelic and genotypic PRNP frequencies, at the three main codons, were determined in four sheep breeds reared in central Italy (Appenninica, Bergamasca, Comisana and Sarda). Moreover homozygous ARQ sheep have been investigated to measure the frequencies of minor allelic variants. In this study the AT112RQ and ARQK176 protective allelic variants were detected. The AT112RQ allele was observed (ranging from 6.3% to 31.8%) in all the analysed breeds with the exception of Sarda sheep. The ARQK176 allele by contrast was observed only in Sarda breed (4.6%). Moreover five non synonymous polymorphisms were also found (Q101R, G127S, L141F, H143R, and H180Y).