Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP^Sc). Disease is transmitted by the autocatalytic propagation of PrP^Sc misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP^Sc generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain‐specific properties. Inoculation of wild‐type mice with in vitro‐generated PrP^Sc caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP^Sc properties.