High temperature is a major environmental factor that limits wheat (Triticum aestivum L.) productivity. Climate models predict greater increases in night-time temperature than in daytime temperature. The objective of this research was to compare the effects of high daytime and high night-time temperatures during anthesis on physiological (chlorophyll fluorescence, chlorophyll concentration, leaf level photosynthesis, and membrane damage), biochemical (reactive oxygen species (ROS) concentration and antioxidant capacity in leaves), growth and yield traits of wheat genotypes. Winter wheat genotypes (Ventnor and Karl 92) were grown at optimum temperatures (25/15 degrees C, maximum/minimum) until the onset of anthesis. Thereafter, plants were exposed to high night-time (HN, 25/24 degrees C), high daytime (HD, 35/15 degrees C), high daytime and night-time (HDN, 35/24 degrees C) or optimum temperatures for 7days. Compared with optimum temperature, HN, HD and HDN increased ROS concentration and membrane damage and decreased antioxidant capacity, photochemical efficiency, leaf level photosynthesis, seed set, grain number and grain yield per spike. Impact of HN and HD was similar on all traits. Greater impact on seed set, grain number and grain yield per spike was observed at HDN compared with HN and HD. These results suggest that HN and HD during anthesis cause damage of a similar magnitude to winter wheat.