Aggregation-prone proteins often misfold into multiple distinct amyloid conformations that dictate different physiological impacts. Although amyloid formation is triggered by a transient nucleus, the mechanism by which an initial nucleus is formed and allows the protein to form a specific amyloid conformation has been unclear. Here we show that, before fiber formation, the prion domain (Sup35NM, consisting of residues 1–254) of yeast prion Sup35, the [PSI⁺] protein determinant, forms oligomers in a temperature-dependent, reversible manner. Mutational and biophysical analyses revealed that 'non-native' aromatic interactions outside the amyloid core drive oligomer formation by bringing together different Sup35NM monomers, which specifically leads to the formation of highly infectious strain conformations with more limited amyloid cores. Thus, transient non-native interactions in the initial nucleus are pivotal in determining the diversity of amyloid conformations and resulting prion strain phenotypes.