Abstract Background Classical scrapie in sheep is caused by several different strains rather than a single strain, as is the case for epidemic classical bovine spongiform encephalopathy (BSE). Polymorphisms R171 and K176 located in the β2-α2 loop region of sheep prion protein (PrP) have been associated with potential protection for the propagation of classical scrapie. Methods The protective role of R171 and K176 polymorphic variants in susceptibility and resistance to different prion strains circulating in Europe was investigated using transgenic mouse lines expressing R171 or K176 sheep PrP in comparable levels (R171-Tg552 and K176-Tg570, respectively). Both lines were intracranially challenged with a panel of isolates representative of diverse prion strains, including at least 4 different classical scrapie strains. These isolates were previously characterized by transmission studies in ovine (Wt-OvPrP-Tg501) and bovine (BoPrP-Tg110) transgenic mice. Results R171-Tg552 and K176-Tg570 mouse lines succumbed after the inoculation of atypical scrapie isolates with 100% attack rates and long survival times. However, the propagation of all tested classical scrapie isolated was completely blocked in R171-Tg552 mice, whereas in K176-Tg570 mice the propagation of most of the classical scrapie isolates was highly restricted or completely blocked depending on the prion strain. BSE transmission to R171-Tg552 mice was only possible after its adaptation to sheep PrP while no infection could be detected in K176-Tg570 mice, even after passage in sheep. Conclusions These results indicate that the R171 and K176 polymorphic variants of the ovine PrP sequence restrict the propagation of prions in a strain-dependent manner and are useful tools for prion strain discrimination.