Stay-green, a key trait of wheat, can not only increase the yield of wheat but also its resistance to heat stress during active photosynthesis. Cytokinins are the most potent general coordinator between the stay-green trait and senescence. The objectives of the present study were to identify and assess the effects of cytokinins on the photosynthetic organ and heat resistance in wheat. Two winter wheat cultivars, Wennong 6 (a stay-green cultivar) and Jimai 20 (a control cultivar), were subjected to heat stress treatment from 1 to 5 days after anthesis (DAA). The two cultivars were sprayed daily with 10 mg L-1 of 6-benzylaminopurine (6-BA) between 1 and 3 DAA under ambient and elevated temperature conditions. We found that the heat stress significantly decreased the number of kernels per spike and the grain yield (P < 0.05). Heat stress also decreased the zeatin riboside (ZR) content, but increased the gibberellin (GA3), indole-3-acetic acid (IAA), and abscisic acid (ABA) contents at 3 to 15 DAA. Application of 6-BA significantly (P < 0.05) increased the grain-filling rate, endosperm cell division rate, endosperm cell number, and 1,000-grain weight under heated condition. 6-BA application increased ZR and IAA contents at 3 to 28 DAA, but decreased GA3 and ABA contents. The contents of ZR, ABA, and IAA in kernels were positively and significantly correlated with the grain-filling rate (P < 0.05), whereas GA3 was counter-productive at 3 to 15 DAA. These results suggest that the decrease in grain yield under heat stress was due to a lower ZR content and a higher GA3 content compared to that at elevated temperature during the early development of the kernels, which resulted in less kernel number and lower grain-filling rate. The results also provide essential information for further utilization of the cytokinin substances in the cultivation of heat-resistant wheat.