Short period of high temperature stress, over 30℃, often happens during grain filling in many wheat production areas in the world, and has been an important factor limiting wheat grain yield and quality. Starch, a major component of wheat grain, has a close link with wheat grain yield and quality. It has been known that the grain starch accumulation reduces due to the high temperature during grain filling, leading to the decreased grain yield. However, there is little knowledge about how the formation of starch components, amylose and amylopectin, and the activity of the related enzymes change when wheat plants are exposed to high temperature stress during grain filling. The study was conducted to investigate the effects of high temperature at different periods of grain filling on grain starch formation of wheat. Two contrasting winter wheat (Triticum aestivum L.) cultivars, JM20 (weak heat tolerance) and LM21 (strong heat tolerance), were grown at Tai’an Experimental Station of Shandong Agriculture University during the 2004-2006 growing season. Two treatments of high temperature stress were made in the field with plastic sheds in the early (5-9d after anthesis, T1) and middle (15-19d after anthesis, T2) grain filling, respectively, in comparison with the control plants that grew naturally. The treatments were arranged in a randomized complete block design with three replications. Effects of high temperature stress at different grain filling periods in field on starch accumulation, starch granule distribution, and activities of the related enzymes were examined in developing grain of winter wheat. The results showed that there was significant effect of high temperature stress on grain starch accumulation. High temperature stress after anthesis remarkably reduced starch accumulation at the maturity. The total starch and amylopectin contents of high temperature treatments decreased markedly, but amylose content of heat treatments increased, as compared with control. Then the ratio of amylose to amylopectin in high temperature treatments was significantly higher than that of control. High temperature stress in the middle period of grain filling had a larger effect on the starch accumulation than that in the early grain filling. The starch accumulation of JM20 was decreased due to high temperature more than that of LM21, indicating that JM20 was more sensitive to high temperature. High temperature resulted in the significant increases of the volume, number and surface area percentage of A type granule, but the decrease of B type granule, compared with control. After 5d high temperature stress, the slight increases occurred in the sucrose content, the activities of sucrose synthase (SS), adnosine diphosphate glucose pyrophosphorylase (AGPP), soluble starch synthase (SSS), granule-bounded starch synthase (GBSS) and starch branching synthase (SBE) in T1 treatments of two cultivars. But after removal of the high temperature stress in T1 treatment, the above parameters in JM20 and LM21 became lower than those of the control at both 15d and 20d. The significant decreases were observed in the sucrose content, SS, AGPP, SSS and SBE activities of T2 treatments, especially in JM20 after 5d high temperature stress. However, only a little difference existed in GBSS activity between high temperature treatment and control. The grain starch accumulation was found in consonance with the grain sucrose content, and the activities of SS, AGPP, SSS, GBSS and SBE, suggesting that it was poor supply of sucrose and the decreased activities of the enzymes involved in starch synthesis that brought about the declined starch accumulation in grain under high temperature stress. As compared with LM21, JM20 had a larger decline in the starch accumulation and the activities of related enzymes, indicating the difference between cultivars existed in tolerance to high temperature. Hence, it is suggested in this paper that growing heat-resistant cultivars, and applying appropriate schedules of irrigation and fertilization would be effective to cut dawn the influence of high temperature stress during grain filling on wheat production.