Human CD1 proteins have long been hypothesized to possess non-redundant biological functions, suggested by their differential tissue distribution, distinct intracellular trafficking pathways, and structurally unique antigen-binding grooves. Our comparative lipidomic platform enables comprehensive profiling of lipid ligands associated with cellular CD1-lipid complexes. Utilizing this advanced approach, we detected more than 300 ion chromatograms that were specifically detected in association with CD1c, including both previously characterized and unknown lipid species that were identified by collisional mass spectrometry. This outcome supports crystallographic data indicating that CD1c, compared to other human isoforms, has a cleft that is characterized by high structural flexibility and diverse lipid capture patterns. Coupling this lipidomic dataset with lipidome-scale T cell receptor (TCR) trapping techniques, we further identified weak and strong CD1c binding motifs that are defined by dual and single alkyl chains, respectively. Further, we present the first analysis of the lipidome of CD1e, which has long been conceived as a soluble lipid transfer protein that does not directly present lipids to T cells. These results point to non-redundant isoform specific lipid capture patterns and provide new perspectives for designing lipids that target individual CD1 proteins.