Isopentenyltransferase (IPT) genes, including those encoding ATP/ADP-IPTs and tRNA-IPTs, control the rate-limiting steps of the biosynthesis of N 6-(Δ 2-isopentenyl)adenine (iP)-type and trans-zeatin (tZ)-type cytokinins and cis-zeatin (cZ)-type cytokinins, respectively. However, the evolution and roles of these IPTs in angiosperms are not well understood. Here, we report comprehensive analyses of the origins, evolution, expression patterns, and possible roles of ATP/ADP-IPTs and tRNA-IPTs in angiosperms. We found that Class I and II tRNA-IPTs likely coexisted in the last common ancestor of eukaryotes, while ATP/ADP-IPTs likely originated from a Class II tRNA-IPT before the divergence of angiosperms. tRNA-IPTs are conservatively retained as 2-3 copies, but ATP/ADP-IPTs exhibit considerable expansion and diversification. Additionally, tRNA-IPTs are constitutively expressed throughout the plant, whereas the expression of ATP/ADP-IPTs is tissue-specific and rapidly downregulated by abiotic stresses. Furthermore, previous studies and our present study indicate that ATP/ADP-IPTs and their products, iPs/tZs, may regulate responses to environmental stresses and organ development in angiosperms. We therefore hypothesize that tRNA-IPTs and the associated cZs play a housekeeping role, whereas ATP/ADP-IPTs and the associated iP/tZ-type cytokinins play regulatory roles in organ development and stress responses in angiosperms, which echoes the conclusions and hypothesis presented in the accompanying study by Wang, X. et al Evolution and roles of cytokinin genes in angiosperms 2: Do ancient CKXs play housekeeping roles while non-ancient CKXs play regulatory roles? Hortic Res