Cytokinins (CKs) function in plant development and during stress responses, but their role in drought tolerance in wheat is unknown. In the present study, 24 isopentenyltransferase (IPT) genes, encoding rate-limiting enzymes in CK biosynthesis were identified in the wheat genome. The chromosomal locations and structures of the genes, protein properties, and phylogenetic relationships were characterized. ATP/ADP TaIPTgenes showed tissue-specific expression. TaIPT2, TaIPT7, and TaIPT8expression was rapidly induced by 0.5–1 h drought treatments, which decreased to low levels after 2 h drought treatment, as did most other TaIPTgenes. TaIPT8-5a/5b/5dtriple mutants showed decreased levels of tZ-type CK under normal and drought conditions and reduced drought tolerance, which, however, did not manifest as phenotype alterations. By contrast, transgenic wheat plants with drought-induced TaIPT8showed increased drought tolerance. Our study provides a foundation for further investigation of TaIPTgenes and novel insights into the role of CKs in the drought response of wheat.