Drought and heat stress episodes are increasing in frequency and severity causing significant crop yield losses. Despite this, these stresses have rarely been studied together in wheat, but their combined effect is known to differ from the effect of each stress singly. To identify physiological mechanisms involved in plant responses and investigate genetic variation, eight wheat accessions were selected from a diverse, worldwide collection for their contrasting tolerance to drought and heat stress. Plants were grown in experiments on an automated gravimetric platform with accurate irrigation that allows the control of water availability and real-time monitoring of transpiration and water use. Drought or combined drought and heat treatments were applied at the early grain filling stage. Grain yield was differentially affected by drought and heat stress depending on the genotype. Interactions between genotype and treatment for aboveground biomass were identified under drought, whereas combined drought and heat treatment differentially affected single grain weight and the water use efficiency of grains. Genotypes contrasted in water use when growing in the same, water-limited conditions following heat stress, as the treatment differentially altered subsequent transpiration response to evaporative demand (vapour pressure deficit). We have investigated whether this genetic variation is related to different spike hydraulic conductance and water potential gradients, isolation and protection of the spike tissues, or water soluble carbohydrates’ allocation in vegetative versus reproductive tissues. The effects of drought and heat cannot be controlled in rainfed wheat and new, more tolerant varieties are required to mitigate adverse effects on productivity. We propose novel physiological traits that can now be measured for drought and heat tolerant wheat.