Chimpanzees represent important models for studying several human pathogens. In the present study, we utilized a combinatorial peptide library to characterize the binding specificities of the chimpanzee class I molecules Patr A*0301 and A*0401, both of which are present in about 17% of chimpanzees. Patr A*0301 was found to recognize peptides using the canonical position 2/C-terminus spacing, with the small residues S, T, and A being the most preferred in position 2, and the positively charged residues R and K preferred at the C terminus. Patr A*0401 was found to recognize a more complex motif where the C terminus and then the residue in positions 1 and/or 5 are the primary anchors. Like A*0301, the C-terminal preference of A*0401 is for positively charged residues. At positions 1 and 5, positively charged and large residues are the most preferred, respectively. Coefficient values derived from the combinatorial library proved to be an efficient means for predicting A*0301 and A*0401 binders. The present data provide detailed information to facilitate the identification of potential T cell epitopes recognized in the context of two common chimpanzee class I alleles, and further validate the combinatorial library approach as an efficient method to characterize class I binding specificities.