Adaptive immunity is initiated by T cell recognition of specific antigens presented by major histocompatibility complexes (MHCs). MHC multimer technology has been developed for the detection, isolation, and characterization of T cells in infection, autoimmunity, and cancer. Here, we present a simple, fast, flexible, and efficient method to generate many different MHC class I (MHC I) multimers in parallel using temperature-mediated peptide exchange. We designed conditional peptides for HLA-A*02:01 and H-2Kb that form stable peptide–MHC I complexes at low temperatures, but dissociate when exposed to a defined elevated temperature. The resulting conditional MHC I complexes, either alone or prepared as ready-to-use multimers, can swiftly be loaded with peptides of choice without additional handling and within a short time frame. We demonstrate the ease and flexibility of this approach by monitoring the antiviral immune constitution in an allogeneic stem cell transplant recipient and by analyzing CD8+ T cell responses to viral epitopes in mice infected with lymphocytic choriomeningitis virus or cytomegalovirus.