Mucosal-Associated Invariant T (MAIT) cells recognize riboflavin pathway-derived metabolites presented by the MHC-I-like molecule MR1. Both MR1 and the T cell receptor genes used by MAIT cells are under strong evolutionary selection in mammals, suggesting important functions across species. As the functional programs of human and mouse MAIT cells appear distinct, the evolutionarily conserved functional features of MAIT cells, if any, remain to be identified. Here, using species-specific MR1 tetramers coupled to single-cell RNA sequencing, we characterized MAIT cell development in 6 species spanning 110 million years of evolution. Human conventional and iNKT cells were also profiled for comparison. Cross-species analyses revealed a conserved sequence of events underlying MAIT cell maturation in the thymus, marked by early expression of ZBTB16 in all species. MAIT cells in human, sheep, cattle and opossum acquired a shared transcriptional program with both type-1 and type-17 components, reflecting ancestral features. This program was also acquired by human iNKT cells, indicating a common differentiation pathway for innate-like T cells. Thymic differentiation led to distinct type-1 and type-17 subsets in rodents, including pet mice and genetically diverse mouse strains, dismissing a laboratory artifacts. However, MAIT-17 cells further matured in mouse intestines to express a remarkably conserved program characterized by concomitant expression of type-1, type-17, cytotoxicity and tissue-repair genes. Altogether, the study defines a deeply conserved transcriptional program for MAIT cells and provides a unifying view on the transcriptional features of innate-like T cells across evolution.