AbstractQuestionWhich optical traits, retrieved from biophysical models applied to Sentinel‐2 images, enable an estimation of tree species diversity based on the Spectral Variation Hypothesis?LocationConiferous mountain forest in the eastern Italian Alps.MethodsWe analyzed the PROSPECT‐5 and Invertible Forest Reflectance Model (INFORM) biophysical parameters as retrieved from canopy reflectance data of different forest plots (using Sentinel‐2 images for the years 2017, 2018 and 2019) as optical trait indicators (OTIs). We successively tested the Spectral Variation Hypothesis (SVH) for each retrieved OTI using the Rao's Q as heterogeneity index, validating them against Shannon's H values calculated as a tree species diversity index derived from in‐situ collected data.ResultsWe demonstrated differences among OTIs in terms of how well their variations can be linked to species diversity. In particular the variations of brown pigments (Cbrown), carotenoids (Car) and chlorophyll content (Cab) can be considered the most relevant OTIs for the application of the SVH when using the Rao's Q as a proxy for tree species diversity in our study site.ConclusionsThis research underlined that the OTIs contribute differently in the SVH to estimate tree species diversity, highlighting significant positive correlations between tree species diversity and the spatial heterogeneity of the estimated pigment content (Cab, Car, Cbrown).