The occurrence and differences of resistant regions containing IgE-binding epitopes of α_S1-casein (α_S1-CN) variants B and C, as well as α_S2-CN A and B, after in vitro gastrointestinal digestion was investigated using mass spectrometry. The amino acid substitutions characterising the genetic variants affected the peptide pattern arising from the caseins and thus modifications in their allergenic epitopes occurred. Peptides f174–193 in α_S1-CN B and f179–198 in α_S1-CN C correspond to the IgE-binding epitope f173–194, which has been reported as one of the major epitopes in α_S1-CN B. Within α_S2-CN, the two variant-specific peptides, f7–29 from variant A and f1–22 from variant B, contain the previously identified IgE-binding epitope f1–20. These peptides, and in consequence the protein variants, may exhibit different immunoreactions, which could be significant in the production of milk with improved nutritional properties, such as hypoallergenic quality, by selection and breeding of cows with particular milk protein genotypes.