Prions are the infectious agents responsible for transmissible spongiform encephalopathy, and are primarily composed of the pathogenic form (PrPSc) of the host-encoded prion protein (PrPC). Recent studies have revealed that protein misfolding cyclic amplification (PMCA), a highly sensitive method for PrPSc detection, can overcome the species barrier in several xenogeneic combinations of PrPSc seed and PrPC substrate. Although these findings provide valuable insight into the origin and diversity of prions, the differences between PrPSc generated by interspecies PMCA and by in vivo cross-species transmission have not been described. This study investigated the histopathological and biochemical properties of PrPSc in the brains of tga20 transgenic mice inoculated with Sc237 hamster scrapie prion and PrPSc from mice inoculated with Sc237-derived mouse PrPSc, which had been generated by interspecies PMCA using Sc237 as seed and normal mouse brain homogenate as substrate. Tga20 mice overexpressing mouse PrPC were susceptible to Sc237 after primary transmission. PrPSc in the brains of mice inoculated with Sc237-derived mouse PrPSc and in the brains of mice inoculated with Sc237 differed in their lesion profiles and accumulation patterns, Western blot profiles, and denaturant resistance. In addition, these PrPSc exhibited distinctive virulence profiles upon secondary passage. These results suggest that different in vivo and in vitro environments result in propagation of PrPSc with different biological properties.