Wheat plants growing under Mediterranean rain-fed conditions are exposed to water deficit, particularly during the grain filling period, and this can lead to a strong reduction in grain yield (GY). This study examines the effects of water deficit after during the grain filling period on photosynthetic and water-use efficiencies at the leaf and whole-plant level for 14 bread wheat genotypes grown in pots under glasshouse conditions. Two glasshouse experiments were conducted, one in a conventional glasshouse at the Universidad de Talca, Chile (Experiment 1), and another at the National Plant Phenomics Centre (NPPC), Aberystwyth, UK (Experiment 2), in 2015. Plants were grown under well-watered (WW) and water-limited (WL) conditions during grain filling. The reductions in leaf water potential (Ψ), net CO2 assimilation (An) and stomatal conductance (gs) due to water deficit were 79, 35 and 55%, respectively, during grain filling but no significant differences were found among genotypes. However, chlorophyll fluorescence parameters (as determined on dark-adapted and illuminated leaves) and chlorophyll content (Chl) were significantly different among genotypes, but not between water conditions. Under both water conditions, An presented a positive and linear relationship with the effective photochemical quantum yield of Photosystem II (Y(II)) and the maximum rate of electron transport (ETRmax), and negative with the quantum yield of non-photochemical energy conversion in Photosystem II (Y(NPQ)). The relationship between An and Chl was positive and linear for both water conditions, but under WL conditions An tended to be lower at any Chl value. Both, instantaneous (An/E) and intrinsic (An/gs) water-use efficiencies at the leaf level exhibited a positive and linear relationship with plant water-use efficiency (WUEp = plant dry weight/water use). Carbon discrimination (Δ13C) in kernels presented a negative relationship with WUEp, at both WW and WL conditions, and a positive relationship with GY. Our results indicate that during grain filling wheat plants face limitations to the assimilation process due to natural senesce and water stress. The reduction in An and gs after anthesis in both water conditions was mainly due a decline in the chlorophyll content (non-stomatal limitation), whereas the observed differences between water conditions were mainly due to a stomatal limitation.