The effects of canopy leaf position, light availability and the presence of fruit on leaf net photosynthesis rate (Pn) were evaluated in a chestnut orchard located in Central Italy. The study was carried out on Castanea sativa Mill. cv. ‘Marrone di Stroncone’, native to the Umbria Region, and on an interspecific hybrid Castanea crenata × Castanea sativa, cv. ‘Marigoule’. ‘Marrone di Stroncone’ had a higher mean Pn than ‘Marigoule’. In both genotypes, Pn changed during the growing season in response to phenological stages and, in particular, was higher during the main fruit growth period. From July to October the part of the shoot with higher Pn moved progressively from the base to the tip. During the day, Pn was high in the morning, and decreased progressively in the afternoon. The nearby presence of fruit increased Pn of leaves on shoots exposed to full sunlight, especially during the morning, but did not significantly influence the leaf chlorophyll (Chl) or carbohydrate contents. Light saturation for Pn was relatively high and there was a drastic reduction in Pn in leaves which, due to their position, intercepted a photosynthetic photon flux density (PPFD) lower than 300–400 μmol photon m−2 s−1 during the day. Shaded leaves were ca. 26% thinner than sunlit ones. Both the epidermal and mesophyll tissues were thicker in sunlit leaves, and the “density” of cells was higher in sunlit leaves. Morpho-anatomical and physiological adaptations allow the chestnut to optimise its use of the limited radiant energy available, but shading greatly reduces productivity. From the results it is affirmed that studies which consider photosynthetic assimilation must take into account the genotype, time of the day, growing season, leaf position and influence of the fruit. In order to increase the efficiency of the tree, it is important to ensure optimal conditions for whole canopy Pn (light interception, water and nutrient availability, leaf integrity, etc.), particularly during those times when demand for assimilates is high. The chestnut genotypes studied have a very low Pn compared to other temperate fruit and nut trees.