In prion diseases, the cellular prion protein PrPC can misfold into PrPSc and auto-organize into conformationally distinct assemblies or strains. A plethora of observations reports the existence of PrPSc structural heterogeneity within prion strains, suggesting the emergence and coevolution of structurally distinct PrPSc subassemblies during prion replication in controlled environment. However, the nature of and the underlying processes of the structural diversification remain poorly explored and mainly based on conjectural observations. Conceptually, such structural heterogeneity appears central to prion host adaptation since the actual paradigm consists mostly on the best replicator selection, i.e. selection of the best-fit subset of PrPSc assemblies within the PrPSc structural cloud. Here, we first characterized the evolution of the PrPSc quaternary structure in early versus late stage of prion replication (in a homotypic PrP context) in vivo and in bona fide cell-free amplification assays. Regardless of the strain studied, the early replication stage conduced to the preferential formation of small PrPSc oligomers, thus highlighting a quaternary structural convergence phenomenon. The evolution of these small oligomeric assemblies led to the formation of structurally different multimers through a secondary templating pathway in an autocatalytic and PrPC-dependent manner. Second, we addressed the role of such structural PrPSc diversification in prion adaptation on cross-species transmission. We isolated by size-fractionation or by dilution the formed PrPSc assemblies and measured their relative propensity to cross an existing species barrier, as modelled by transgenic mice expressing heterotypic PrPC. We showed impaired and aberrant fitness of the isolated PrPSc subassemblies as compared to the unfractionated and undiluted populations, respectively. Altogether our observations uncover the existence of two mechanistic processes during prion replication: first, prion structural diversification occurs through a secondary templating pathway. Second, overcoming species barriers requires complementation between the PrPSc subassemblies. These processes explain how structurally diverse assemblies can coexist despite competition for the same PrPC substrate and truly challenge the best- replicator selection models.