The HLA-B*27 peptidome has drawn significant attention due to the genetic association between some of the HLA-B*27 alleles and the inflammatory rheumatic disease ankylosing spondylitis (AS). The role of HLA-B*27 in this condition is not known yet. This study aims to expand the known limits of the HLA-B*27 peptidome in order to facilitate selection and testing of new peptides, possibly implicated with the disease. The HLA peptidomes of HeLa and C1R cell lines stably transfected with the AS-associated HLA-B*27:05 allele, the non-associated HLA-B*27:09 allele, or their mutants with Cysteine 67 replaced by Serine (C67S), were analyzed on a very large scale. In addition, the peptidomes of HLA-B*27:05 and HLA-B*27:05-C67S were analyzed from transgenic rats’ spleens. The results indicate that a fraction of HLA-B*27 peptides contain lysine at their second position (P2), in addition to the more commonly found peptides with arginine, or the less common glutamine located at this anchor position. Furthermore, the C67S mutation increases the percentages of peptides with glutamine or lysine at their P2 position, in both HLA-B*27:05 and HLA-B*27:09. Therefore, peptides with P2 lysine should be considered as valid ligands of HLA-B*27 molecules and taken into account while looking for candidate for arithrogenic peptides.