Prions are infectious proteins composed of the abnormal disease-causing isoform PrP(Sc), which induces conformational conversion of the host-encoded normal cellular prion protein PrP(C) to additional PrP(Sc). The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrP(Sc) with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrP(C), strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrP(C). The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrP(Sc) conformational compatibility, determines prion strain mutation in these cervids.