DQα and DQβtrans heterodimeric HLA‐DQ molecules form in individuals heterozygous for the DQ2 and DQ8 specificities. Unique functions and disease associations have been postulated for such trans‐dimers, which may be different from cis‐encoded DQ molecules encoded by the corresponding haplotypes. We analyzed the ability of the trans‐dimer encoded by HLA‐DQA1*0501/DQB1*0302 to bind a peptide antigen which interacts with DQ molecules encoded by both parental haplotypes. Markedly impaired binding was observed, consistent with both the use of different anchor residues and with changes in levels of DQ cis‐dimer availability for peptide binding interactions.