Wheat (Triticum aestivum) is one of the world’s most important cereal crops, with more than 700 million tons produced each year. As a result of climate change, drought is one of the major abiotic stresses constraining crop productivity worldwide. With the need to ensure food security and increase crop productivity in challenging environments, there is a desire to identify and understand the genetic mechanisms underlying drought tolerance in wheat, and utilise these in the development of novel, drought tolerant varieties. To identify the developmental stage most impacted by drought, we profiled the transcriptomic response of the elite cultivar Paragon to water deficit. Drought was induced at six developmental stages, following the Zadoks’ scale. Physiological measurements were made and tissue harvested for RNA extraction and biochemical assays. The impact of water deficit at each specific growth stage, was also assessed by calculating final biomass and yield after re-watering. Physiological measurements indicated that for the wheat cultivar Paragon, the productivity of glasshouse-grown plants is most affected when water deficit is induced at flowering (Zadoks stage 6). mRNA-seq analysis enabled us to identify genes transiently differentially expressed across these developmental stages during periods of water deficit. Extensive variability for drought tolerance has been observed within wheat wild relatives and landraces. In collaboration with University of Lancaster, we also identified a bread wheat landrace cultivar from the Watkins Core Collection with > 1.5 times water use efficiency compared to the elite cultivar Paragon. To further characterise this drought tolerance, we compared well-watered plants to those exposed to a water deficit at the reproductive growth stages Z5-6, for both Paragon and the landrace cultivar. Pairwise comparisons of the transcriptomes allowed us to identify differentially expressed genes associated with differences in cultivar and drought treatment applied. We plan to use these results to characterise the genetic mechanisms, and regulatory networks underlying increased water use efficiency and drought tolerance in wheat.