This study attempts (1) to evaluate the capability of hyperspectral reflectance to differentiate C 3 and C 4 grass species, both in isolation and in mixed canopies; (2) to identify the critical spectral ranges that differentiate the two groups and individual species within them; and (3) to determine if there is temporal variation in these capabilities. During one year, hyperspectral reflectance of C 3 and C 4 grass species was measured both in single-species and in mixed canopies. Spectral bands with higher differentiating potential were identified and species classified. For singlespecies canopies, hyperspectral reflectance differentiated the two functional groups and most species in all seasons. In mixed canopies, it underestimated the fractional cover of the C 4 component. The green, red, and near infrared above 820 nm spectral ranges were critical both for species and functional group differentiation. In conclusion, hyperspectral information was useful to differentiate pure canopies, but the differentiation algorithms were season-specific. Additionally, we need to improve our understanding of interactive effects of species in order to accurately estimate the composition of assemblages.