Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele‐specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II‐bound peptides and found that HLA‐DQ2‐eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA‐DPw3/DPw4‐eluted peptide pool yielded a sequence matching an epitope from the endogeneous enzyme glyceraldehyde‐3‐phosphate dehydrogenase. This self‐peptide and a partially identical, known allo‐epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II‐eluted peptides.