A significant fraction of CD1d‐restricted T cells express an invariant T cell receptor (TCR) α‐chain. These highly conserved invariant NKT (iNKT) populations are important regulators of a wide spectrum of immune responses. The ability to directly identify and manipulate iNKT cells is essential to understanding their function and to exploit their therapeutic potential. To this end, we sought monoclonal and polyclonal antibodies specific for iNKT cells by immunizing CD1d KO mice, which lack iNKT cells, with a cyclic peptide modeled after the TCRα CDR3 loop. One mAb (6B11) was specific for cloned and primary human but not rodent iNKT cells and the human invariant TCRα, as shown by transfection and reactivity with human invariant TCRα transgenic T cells ex vivo and in situ. 6B11 was utilized to identify, purify, and expand iNKT cells from an otherwise minor component of human peripheral blood lymphocytes and to specifically identify human iNKT cells in tissue. Thus, we report a novel and general strategy for the generation of mAb specific for the CDR3 loop encoded by the TCR of interest. Specifically, an anti‐Vα24Jα18 CDR3 loop clonotypic TCR mAb is available for the enumeration and therapeutic manipulation of human and non‐human primate iNKT populations.