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In this study, daily changes over a short period and diurnal progression of spectral reflectance at the leaf level were used to identify spring wheat genotypes ( Triticum aestivum L.) susceptible to adverse conditions. Four genotypes were grown in pots experiments under semi-controlled conditions in Chile and Spain. Three treatments were applied: i) control ( C ), ii) water stress ( WS ), and iii) combined water and heat shock ( WS+T ). Spectral reflectance, gas exchange and chlorophyll fluorescence measurements were performed on flag leaves for three consecutive days at anthesis. High canopy temperature ( H CT ) genotypes showed less variability in their mean spectral reflectance signature and chlorophyll fluorescence, which was related to weaker responses to environmental fluctuations. While low canopy temperature ( L CT ) genotypes showed greater variability. The genotypes spectral signature changes, in accordance with environmental fluctuation, were associated with variations in their stomatal conductance under both stress conditions ( WS and WS+T ); L CT genotypes showed an anisohydric response compared that of H CT , which was isohydric. This approach could be used in breeding programs for screening a large number of genotypes through proximal or remote sensing tools and be a novel but simple way to identify groups of genotypes with contrasting performances., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Estrada, Flexas, Araus, Mora-Poblete, Gonzalez-Talice, Castillo, Matus, Méndez-Espinoza, Garriga, Araya-Riquelme, Douthe, Castillo, del Pozo and Lobos.)