Prion diseases are caused by a misfolded isoform of the prion protein, PrPSc. Prion strains are hypothesized to be encoded by strain-specific conformations of PrPSc and prions can interfere with each other when a long-incubation period strain (i.e. blocking strain) inhibits the conversion of a short-incubation period strain (i.e. non-blocking). Prion strain interference influences prion strain dynamics and the emergence of a strain from a mixture; however, it is unknown if two long-incubation period strains can interfere with each other. Here, we show that co-infection of animals with combinations of long-incubation period strains failed to identify evidence of strain interference. To exclude the possibility that this inability of strains to interfere in vivo was due to a failure to infect common populations of neurons we used protein misfolding cyclic amplification strain interference (PMCAsi). Consistent with the animal bioassay studies, PMCAsi indicated that both co-infecting strains were amplifying independently, suggesting that the lack of strain interference is not due to a failure to target the same cells but is an inherent property of the strains involved. Importantly PMCA reactions seeded with long incubation-period strains contained relatively higher levels of remaining PrPC compared to reactions seeded with a short-incubation period strain. Mechanistically, we hypothesize the abundance of PrPC is not limiting in vivo or in vitro resulting in prion strains with relatively low prion conversion efficiency to amplify independently. Overall, this observation changes the paradigm of the interactions of prion strains and has implications for interspecies transmission and emergence of prion strains from a mixture.