Detection of grassland canopy chlorophyll fluorescence (Chl-F) conducted with an imaging spectroradiometer provided evidence of potential remote sensing estimation of steady-state Chl-F (Chl-F_s). Daily near-nadir views of extremely high spatial resolution hyperspectral images were acquired from a distance of 4 m for temperate montane grassland in the Czech Republic. Simultaneously, measurements of Chl-F and total chlorophyll content (Chl_a + b) were made on a single leaf at ground level were collected. A specifically designed ‘shade removal’ experiment revealed the influence of dynamic physiological plant processes on hyperspectral reflectance of three wavelengths: 532, 686 and 740 nm. Based on this information, the vegetation indexes R₆₈₆/R₆₃₀, R₇₄₀/R₈₀₀ and PRI calculated as (R₅₃₂–R₅₇₀)/(R₅₃₂+R₅₇₀) were tested for statistical significance with directly measured Chl-F parameters (maximum fluorescence yield, F_v/F_m; steady-state chlorophyll fluorescence, Chl-F_s and actual quantum yield, Ф_II). The grassland species under investigation were: Festuca rubra agg. (L.), Hieracium sp., Plantago sp., Nardus stricta (L.) and Jacea pseudophrygia (C.A. Meyer). The coefficients of determination (R²) for best-fit relationships between PRI-Ф_II and PRI-Chl-F_s, measured in the daily course, show a high variability of 0.23–0.78 and 0.20–0.65, respectively. Similarly, R² for the R₆₈₆/R₆₃₀-Ф_II and R₆₈₆/R₆₃₀-Chl-F_s relationships varied between 0.20–0.73 and 0.41–0.70, respectively. The highest average R² values were found between PRI and Chl_a + b (0.63) and R₆₈₆/R₆₃₀ and Chl_a + b (0.72). The ratio R₇₄₀/R₈₀₀ did not yield a statistically significant relation with Chl-F parameters.