Human T cells evolved to recognize peptide and non-peptide antigens produced by pathogens such as Mycobacterium tuberculosis (M.tb). The human genome contains five CD1 genes that vary in the types of lipids they bind as well as their patterns of cellular expression and intracellular trafficking. We conducted an evolutionary genetic analysis of CD1 genes in humans and non-human primates. Apart from CD1A in olive baboon, each paralog exists as a single gene copy in each primate species and forms a phylogenetically distinct group that generally matches the species phylogeny. These data suggest a lack of diversifying selection over 50 million years of evolution, which stands in stark contrast to the history of the major histocompatibility complex system for presenting peptide antigens to T cells. We found evidence of positive selection in CD1A, CD1C, and CD1E, and such rapidly evolving sites overwhelmingly clustered in the ligand-binding groove. Because CD1D was the most conserved antigen-presenting molecule, we next explored whether molecular interactions between CD1D and T-cell receptors (TCR) were also conserved across species. We used human CD1D tetramers loaded with α-galactosylceramide (α-GalCer) to sort invariant NKT (iNKT) cells from a healthy rhesus macaque. We derived an iNKT-cell line, which bound the tetramer with high avidity and produced IFN-ɣ and TNF-α when restimulated with α-GalCer-loaded human CD1D-transfectants. The dominant TCR-α and TCR-β chains of the rhesus iNKT cell line showed high homology to the human iNKT TCR, including residues known to be critical for binding CD1D-α-GalCer. These data show that the interaction of the iNKT TCR and CD1D-α-GalCer is nearly uniform between humans and non-human primates. Our genetic analysis and molecular studies also provide proof-of-concept that human CD1 tetramers can be repurposed to probe the basic biology of CD1-restricted T-cells in non-human primate models of disease.