Major histocompatibility complex class I-like CD1 molecules have evolved to present lipid-based antigens to T cells. Differences in the antigen-binding clefts of the CD1 family members determine the conformation and size of the lipids that are presented, although the factors that shape CD1 diversity remain unclear. In mice, two homologous genes, CD1D1 and CD1D2, encode the CD1d protein, which is essential to the development and function of Natural Killer T (NKT) cells. However, it remains unclear whether both CD1d isoforms are equivalent in their antigen presentation capacity and functions. CD1d2 molecules can be expressed in the thymus of some mouse strains where they select functional type I NKT cells. Intriguingly, the TCR repertoire and phenotype of CD1d2-selected type I iNKT cells in CD1D1^-/- mice differs from CD1d1-selected iNKT cells. The structure of CD1d2 revealed that the A´ pocket of its antigen-binding groove is markedly restricted in size compared to CD1d1 molecules. Accordingly, CD1d2 molecules cannot present glycolipid antigens with long acyl chains efficiently, favoring the presentation of short acyl chains antigens. These results indicate that the two CD1d molecules present different sets of self-antigen(s) in the mouse thymus, thereby potentially impacting the development of iNKT cells.