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It is widely known that prion strains can mutate in response to modification of the replication environment and we have recently reported that prion mutations can occur in vitro during amplification of vole-adapted prions by Protein Misfolding Cyclic Amplification on bank vole substrate (bvPMCA). Here we exploited the high efficiency of prion replication by bvPMCA to study the in vitro propagation of natural scrapie isolates. Although in vitro vole-adapted PrPSc conformers were usually similar to the sheep counterpart, we repeatedly isolated a PrPSc mutant exclusively when starting from extremely diluted seeds of a single sheep isolate. The mutant and faithful PrPSc conformers showed to be efficiently autocatalytic in vitro and were characterized by different PrP protease resistant cores, spanning aa ∼155–231 and ∼80–231 respectively, and by different conformational stabilities. The two conformers could thus be seen as different bona fide PrPSc types, putatively accounting for prion populations with different biological properties. Indeed, once inoculated in bank vole the faithful conformer was competent for in vivo replication while the mutant was unable to infect voles, de facto behaving like a defective prion mutant. Overall, our findings confirm that prions can adapt and evolve in the new replication environments and that the starting population size can affect their evolutionary landscape, at least in vitro. Furthermore, we report the first example of “authentic” defective prion mutant, composed of brain-derived PrPC and originating from a natural scrapie isolate. Our results clearly indicate that the defective mutant lacks of some structural characteristics, that presumably involve the central region ∼90–155, critical for infectivity but not for in vitro replication. Finally, we propose a molecular mechanism able to account for the discordant in vitro and in vivo behavior, suggesting possible new paths for investigating the molecular bases of prion infectivity., Author Summary Prions are unique infectious agents, consisting of PrPSc, a self-propagating aggregated conformer of the host-encoded prion protein PrPC. Despite the absence of any nucleic acid information, prions exist as distinct strains that share the same amino acid sequence but differ in their conformation. Moreover, prions can mutate and are thus heterogeneous populations able to evolve and adapt to new replication environments. During in vitro amplification of sheep scrapie, we found that a prion mutant could be obtained from one natural isolate. The prion mutant identified was characterized in vivo and in vitro, showing unusual biochemical and biological features: a smaller than usual C-terminal proteinase resistant core of PrPSc, which spans aa ∼155–231, and the inability to propagate in vivo despite an efficient autocatalytic replication in vitro. With such a signature, we denoted the mutant as a “defective” prion mutant. We thus postulate a new hypothesis for the discrepancy between the in vitro and in vivo behavior of the defective mutant and suggest that the central PrPSc domain ∼90–160 might have a key role in prion replication. This work provides important new insights into the mechanism underpinning prion replication and has numerous implications for understanding the molecular requirements indispensable for prion infectivity.