Plant species diversity is an important metric of ecosystem functioning, but field assessments of diversity are constrained in number and spatial extent by labor and other expenses. We tested the utility of using spatial heterogeneity in the remotely-sensed reflectance spectrum of grassland canopies to model both spatial turnover in species composition and abundances (β diversity) and species diversity at aggregate spatial scales (γ diversity). Shannon indices of γ and β diversity were calculated from field measurements of the number and relative abundances of plant species at each of two spatial grains (0.45 m2 and 35.2 m2) in mesic grasslands in central Texas, USA. Spectral signatures of reflected radiation at each grain were measured from ground-level or an unmanned aerial vehicle (UAV). Partial least squares regression (PLSR) models explained 59–85% of variance in γ diversity and 68–79% of variance in β diversity using spatial heterogeneity in canopy optical properties. Variation in both γ and β diversity were associated most strongly with heterogeneity in reflectance in blue (350–370 nm), red (660–770 nm), and near infrared (810–1050 nm) wavebands. Modeled diversity was more sensitive by a factor of three to a given level of spectral heterogeneity when derived from data collected at the small than larger spatial grain. As estimated from calibrated PLSR models, β diversity was greater, but γ diversity was smaller for restored grassland on a lowland clay than upland silty clay soil. Both γ and β diversity of grassland can be modeled by using spatial heterogeneity in vegetation optical properties provided that the grain of reflectance measurements is conserved.