MHC class I molecules display peptides at the cell surface to cytotoxic T cells. The co-factor tapasin functions to ensure that MHC I becomes loaded with high affinity peptides. In most mammals, the tapasin gene appears to have little sequence diversity and few alleles and is located distal to several classical textitMHC I loci, so tapasin appears to function in a universal way to assist MHC I peptide loading. In contrast, the chicken tapasin gene is tightly linked to the single dominantly expressed textitMHC I locus and is highly polymorphic and moderately diverse in sequence. Therefore, tapasin-assisted loading of MHC I in chickens may occur in a haplotype-specific way, via the co-evolution of chicken tapasin and MHC I. Here we demonstrate a mechanistic basis for this co-evolution, revealing differences in the ability of two chicken MHC I alleles to bind and release peptides in the presence or absence of tapasin, where, as in mammals, efficient self-loading is negatively correlated with tapasin-assisted loading. We found that a polymorphic residue in the MHC I α3 domain thought to bind tapasin influenced both tapasin function and intrinsic peptide binding properties. Differences were also evident between the MHC alleles in their interactions with tapasin. Last, we show that a mismatched combination of tapasin and MHC alleles exhibit significantly impaired MHC I maturation in vivo and that polymorphic MHC residues thought to contact tapasin influence maturation efficiency. Collectively, this supports the possibility that tapasin and BF2 proteins have co-evolved, resulting in allele-specific peptide loading in vivo. Tapasin edits the MHC I peptide repertoire and is highly polymorphic in birds but not mammals.Results: Two chicken MHC I alleles differ in peptide binding properties and participate in an allele-specific interaction with tapasin.Conclusion: Tapasin-MHC alleles have co-evolved by balancing interaction characteristics against MHC peptide-binding ability.Significance: Variations in the functional attributes of tapasin and MHC I alleles determine effective antigen presentation.