Heat stress frequently imposes a strong negative impact on vegetative and reproductive development of plants leading to severe yield losses. Wheat, a major temperate crop, is more prone to suffer from increased temperatures than most other major crops. With heat waves becoming more intense and frequent, as a consequence of global warming, a decrease in wheat yield is highly expected. Here, we examined the impact of a short-term (48 h) heat stress on wheat imposed during reproduction at the pollen mitosis stage both, at the physiological and molecular level. We analyzed two sets of summer wheat germplasms from Australia (Kukri, Drysdale, Gladius, and RAC875) and Europe (Epos, Cornetto, Granny, and Chamsin). Heat stress strongly affected gas exchange parameters leading to reduced photosynthetic and transpiration rates in the European cultivars. These effects were less pronounced in Australian cultivars. Pollen viability was also reduced in all European cultivars. At the transcriptional level, the largest group of heat shock factor genes (type A HSFs), which trigger molecular responses as a result of environmental stimuli, showed small variations in gene expression levels in Australian wheat cultivars. In contrast, HSFs in European cultivars, including Epos and Granny, were strongly downregulated and partly even silenced, while the high-yielding variety Chamsin displayed a strong upregulation of type A HSFs. In conclusion, Australian cultivars are well adapted to moderate heat stress compared to European summer wheat. The latter strongly react after heat stress application by downregulating photosynthesis and transpiration rates as well as differentially regulating HSFs gene expression pattern.