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One way to increase yield potential in wheat is screening for natural variation in photosynthesis. This study uses measured and modelled physiological parameters to explore genotypic diversity in photosynthetic capacity (P c, Rubisco carboxylation capacity per unit leaf area at 25 °C) and efficiency (P eff, P c per unit of leaf nitrogen) in wheat in relation to fertilizer, plant stage, and environment. Four experiments (Aus1, Aus2, Aus3, and Mex1) were carried out with diverse wheat collections to investigate genetic variation for Rubisco capacity (V cmax25), electron transport rate (J), CO2 assimilation rate, stomatal conductance, and complementary plant functional traits: leaf nitrogen, leaf dry mass per unit area, and SPAD. Genotypes for Aus1 and Aus2 were grown in the glasshouse with two fertilizer levels. Genotypes for Aus3 and Mex1 experiments were grown in the field in Australia and Mexico, respectively. Results showed that V cmax25 derived from gas exchange measurements is a robust parameter that does not depend on stomatal conductance and was positively correlated with Rubisco content measured in vitro. There was significant genotypic variation in most of the experiments for P c and P eff. Heritability of P c reached 0.7 and 0.9 for SPAD. Genotypic variation and heritability of traits show that there is scope for these traits to be used in pre-breeding programmes to improve photosynthesis with the ultimate objective of raising yield potential. [ABSTRACT FROM AUTHOR]