A predominant system of multiple MHC class I-like restricted innate-like T cells have been identified in Xenopus laevis tadpoles. Using a combination of different reverse genetic approaches, we have identified two, functionally distinct iT cell populations (iVα6 and Vα45) restricted by the Xenopus MHC class I-like genes XNC10 and XNC4, respectively. While iVα6 T cells are critical for anti-ranaviral immunity, iVα45 T cells are dispensable for ranaviral immunity but required for anti-mycobacterial immune defense. Using Xenopus XNC10 tetramers (XNC10-T) we have previously reported the isolation of two populations of iVα6 T cells reminiscent of type I and type II iNKT cells. Here, we successfully generated XNC4 tetramers (XNC4-T) that bind to a distinct but more heterogeneous population of T cells than XNC10-T. Notably, TCRα repertoire analysis of XNC4 tetramer positive (cells sorted from either the spleen or the liver during early (6 days) and late (36 days) stages following Mycobacterium marinum (Mm) infections exhibited different bias. As expected XNC4-T sorted cells from the early stage of Mm infection showed a predominant usage of Va45. In contrast, XNC4-T positive cells isolated from late stage Mm infection did not express Vα45 but did rather displayed a highly biased TCRα repertoire with different dominant Vα segments in infected spleen and liver. These data suggest that MHC class I-like XNC4 may interact with different innate-like or invariant T cell effectors.