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Accurate understanding of the variability in foliar physiological traits across landscapes is critical to improve parameterization and evaluation of terrestrial biosphere models (TBMs) that seek to represent the response of terrestrial ecosystems to a changing climate. Numerous studies suggest imaging spectroscopy can characterize foliar biochemical and morphological traits at the canopy scale, but there is only limited evidence for retrieving canopy photosynthetic capacity (e.g., maximum carboxylation rate, V c,max and maximum electron transport rate, J max). Moreover, the effect of canopy structure within forest communities on scaling up spectra-trait relationships from leaf to canopy level is not well known. To advance the spectra-trait approach and enable the estimation of key traits using remote sensing, we collected imaging spectroscopy data from an Unoccupied Aerial System (UAS) platform over two forest sites in China (a subtropical forest in Mt. Dinghu and a tropical rainforest in Xishuangbanna). At these sites, we also collected ground measurements of leaf spectra and traits, including biochemical (leaf nitrogen, phosphorus, chlorophyll, and water content), morphological (leaf mass per area, LMA) and physiological (V c,max25 and J max25) traits (n = 135 tree-crowns from 42 species across two sites). Using a partial least-squares regression (PLSR) approach, we built and tested spectra-trait models with repeated cross-validation. The spectral models developed with leaf spectra were directly transferred to canopy spectra to evaluate the effect of canopy structure. We further applied canopy spectral models to map these traits at individual tree-crown scale. The results demonstrate that (1) UAS-based canopy spectra can be used to estimate V c,max (R 2 = 0.55, nRMSE = 11.79%), J max (R 2 = 0.54, nRMSE = 12.34%), and five additional foliar traits (R 2 = 0.38–0.60, nRMSE = 10.11–13.56%) at the tree-crown scale with demonstrated generalizability across two sites; (2) canopy structure strongly affects the spectra-trait relationships from leaf to canopy level, but the effects vary considerably across foliar traits and cannot be well captured by the 4SAIL canopy radiative transfer model. UAS-based imaging spectroscopy maps large variability in all foliar traits (including physiological traits) with spatially explicit information, reproducing the field-observed inter- and intra-specific variations. These results demonstrate the capability of using UAS-based imaging spectroscopy for characterizing the variability of foliar physiological traits at individual tree-crown scale over forest landscapes and highlight the similar generalizability but different biophysical mechanisms underlying spectra-trait relationships at leaf and canopy levels. • UAS-based imaging spectroscopy well estimates canopy foliar photosynthetic capacity. • Canopy structure strongly affects the canopy-scale spectra-trait relationships. • UAS imaging spectroscopy maps trait variations at inter- and intra-specific levels. [ABSTRACT FROM AUTHOR]