In this study we extend tetramerization technology to T-cell receptors (TCRs). We identified TCR alpha beta pairs in the absence of accessory molecules, ensuring isolation of high-affinity TCRs that maintain stable binding characteristics after tetramerization. Subtle changes in cognate peptide levels bound to the class I molecule were accurately reflected by parallel changes in the mean fluorescence intensity of cells that bound TCR tetramers, allowing us to accurately assess the binding affinity of a panel of peptides to major histocompatibility complex (MHC) class I. Using a TCR tetramer specific for the Mamu-A(*)01 allele, we identified animals expressing this restricting class I allele from a large cohort of outbred rhesus macaques. TCR tetramers should facilitate analysis of the MHC-peptide interface and, more generally, the design of immunotherapeutics and vaccines.