High temperature is a major environmental factor limiting wheat (Triticum aestivum L.) productivity. Climate models predict greater increase in nighttime temperature compared to daytime temperature. Understanding of the differential responses of wheat plants to high daytime or nighttime temperatures is needed to anticipate the impacts of climate change on wheat production, and to develop appropriate management practices.  The objectives of this research were to quantify and compare the effects of high day- and/or nighttime temperatures during anthesis on physiological (chlorophyll a fluorescence, chlorophyll content, leaf level photosynthesis, and membrane damage), biochemical (reactive oxygen species content, and total 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°C, maximum/minimum) until the onset of anthesis. Thereafter, plants were exposed to high nighttime (HN, 25/24°C), high daytime (HD, 35/15°C), high day- and nighttime (HDN, 35/24°C), or optimum temperatures for 7 days. Compared to optimum temperature, HN, HD, and HDN increased reactive oxygen species content and membrane damage, and decreased antioxidant capacity, photochemical efficiency, and leaf level photosynthesis. All temperature treatments (HN, HD, and HDN) decreased grain weight through decreased grain number and seed set. Impact of HN and HD was similar on most traits including seed set, grain number, and grain weight. Greater impact on seed set, grain number, and grain weight was observed at HDN compared to HN and HD. These results suggest that HN and HD during anthesis cause similar magnitude of damage to winter wheat. This study emphasizes the need for genotypes that are tolerant to high temperature stress.