The association of HLA-B27 with spondyloarthropathy is one of the strongest HLA-associations documented for any autoimmune disease. A common hypothesis for this association is the arthritogenic peptide concept. This dictates that differences in the peptide binding preferences of disease associated and non-associated HLA-B27 allotypes underpin the presentation of bacterial and self-peptides leading to cross-reactive T cell immunity and subsequent autoimmune attack of affected tissues. We have examined qualitative differences in the peptides bound to the 8 most frequent HLA-B27 subtypes by tandem mass spectrometry and quantitative changes in allelic binding specificities determined by highly sensitive and targeted multiple reaction monitoring mass spectrometry. Here we identify over 7500 MHC class I peptides derived from the 8 most common HLA-B27 allotypes, HLA-B*27:02 to HLA-B*27:09 under normal conditions and during infection with Salmonella. Unexpectedly, the bound peptide repertoires of these closely related alleles show significant overlap, with allelic polymorphism resulting in changes in the amino acid composition of the antigen binding cleft manifests largely as quantitative changes in the peptide cargo of these molecules. Thus, absolute binding preferences do not explain disease association. The arthritogenic peptide theory needs to be reassessed in terms of quantitative changes in self-peptide presentation, altered conformation of bound peptides and the propensity of HLA B27 to misfold and form novel immunogenic structures during infection and ER stress.