The coding sequence of the prion gene (PRNP) is highly conserved in mammals however, an elevated number of polymorphisms has been reported in this gene in goats and sheep. Specific genetic variants in the PRNP coding region have been correlated with susceptibility/resistance to the onset of scrapie in sheep but atypical cases have been reported in several breeds, suggesting additional mutations may functionally affect this gene. Fragments from the PRNP promotor, coding region and 3’-UTR spanning approximately 1850bp were resequenced in a total of 96 animals from 13 different locally adapted and commercial sheep breeds from Brazil to prospect novel SNPs. Sequence analysis identified five previously unreported polymorphisms: one in the Promoter region, two in the coding sequence and two in the 3’-UTR. In addition, 27 previously reported SNPs were observed. All of the SNPs identified in the coding region result in predicted amino acid substitutions. LD analysis revealed two regions in strong disequilibrium (D’ values between 0,94 and 1) in the promoter and the 3’-UTR regions. A total of 29 SNPs in the PRNP gene, and an additional 67 SNPs from flanking regions were used to design a Golden Gate assay to genotype sheep samples from different breeds sampled in Brazil. Provisional data analysis revealed LD patterns consistent with results previously obtained with genotyping for polymorphisms in codons 136, 154, 171.