The primary function of MHC-E-human leukocyte antigen (HLA)-E in humans and Mamu-E in rhesus macaques-relates to immune surveillance via CD94/NKG2x receptors expressed on NK cells. However, a secondary role where MHC-E presents immunogenic peptides to CD8+ T cells that provide protective immunity in specific settings has also been described. Given the high sequence homologies between HLA-E and Mamu-E molecules, peptide binding similarities are assumed but not systematically explored, with most studies prioritising HLA-E. Here, we have optimised and developed two complementary techniques to explore the peptide repertoires of specific HLA-E and Mamu-E subtypes. We established a label-free, high-throughput nano-differential scanning fluorimetry (nDSF)-based method, where peptide binding strength is measured through thermal stability (Tm). This method revealed shared repertoires with occasional subtype-specific peptide binding hierarchies for the MHC-E types studied here, HLA-E*01:03 and Mamu-E*02:04. When combined with a fluorescence polarisation (FP) peptide competition assay, we show that half maximal inhibitory concentrations (IC50) correlate exponentially with nDSF-acquired Tm data, revealing that modest Tm increments equate to marked IC50 differences, and hence substantial differences in relative peptide binding strengths. Collectively, these methodologies offer high-throughput, scalable approaches to provide detailed peptide binding information for rhesus and human MHC-E types.