Amino acid polymorphisms of the prion protein gene influence sheep susceptibility to classical and atypical scrapie. Substitutions at codons 136, 154 and 171 play an important role in classical scrapie. Codon 141 leucine to phenylalanine mutation (AFRQ) has been recognized as an increased risk factor for atypical scrapie. In addition a rare allele with lysine at codon 171 (ARK) has been detected in Mediterranean sheep breeds. The presence of ARK poses two problems: the determination of its frequency and its possible interference with genotyping output of routine methods lacking specific detection capacity for ARK. The aim of our work was the development of a routine genotyping method with the capacity to identify ARK and AFRQ in addition to the normally detected alleles and to determine the frequencies of all these alleles in 5 main Italian breeds: Sarda (n=2494), Bergamasca (n=2686), Appenninica (n=297), Comisana (n=361) and Massese (n=402). A multiplex primer extension assay targeting the six single nucleotide polymorphisms of interest was developed. Allele frequencies revealed a very low level of ARR in Bergamasca (6.91%) as opposed to the other breeds, very diverse levels of AFRQ ranging from absence in Comisana to 10.70% in Massese and a restricted presence of ARK. This allele has only been detected in Bargamasca with a significant 3.67% and marginally in Appenninica (0.34%). These results underline the need for adequate routine methods for genotyping of breeds with alleles that can interfere with typing of important codons such as the case of ARK for codon 171.