High temperature stress is an important abiotic stress limiting sorghum (Sorghum bicolor (L.) Moench) yield in arid and semiarid regions. Climate models project greater increases in the magnitude of night temperature compared with day temperature. We hypothesise that high night temperature (HNT) during flowering will cause oxidative damage in leaves and pollen grains, leading to decreased photosynthesis and seed-set, respectively. The objectives of this research were to determine effects of HNT on (1) photochemical efficiency and photosynthesis of leaves, and (2) pollen functions and seed-set. Sorghum plants (hybrid DK-28E) were exposed to optimum night temperature (ONT; 32:22°C, day maximum: night minimum) or HNT (32:28°C, day maximum:night minimum) for 10 days after complete panicle emergence. Exposure to HNT increased thylakoid membrane damage and non-photochemical quenching. However, HNT decreased chlorophyll content, quantum yield of PSII, photochemical quenching, electron transport rate and photosynthesis of leaves as compared with ONT. Exposure to HNT increased the reactive oxygen species (ROS) level of leaves and pollen grains. Lipid molecular species analyses in pollen grains showed that HNT decreased phospholipid saturation levels and altered various phospholipid levels compared with ONT. These changes in phospholipids and greater ROS in pollen grains may be responsible for decreased pollen function, leading to lower seed-set.