Quantitative, spatially explicit estimates of canopy nitrogen are essential for understanding the structure and function of natural and managed ecosystems. Methods for extracting nitrogen estimates via hyperspectral remote sensing have been an active area of research. Much of this research has been conducted either in the laboratory, or in relatively uniform canopies such as crops. Efforts to assess the feasibility of the use of hyperspectral analysis in heterogeneous canopies with diverse plant species and canopy structures have been less extensive. In this study, we use in situ and aircraft hyperspectral data to assess several empirical methods for extracting canopy nitrogen from a tallgrass prairie with varying fire and grazing treatments. The remote sensing data were collected four times between May and September in 2011, and were then coupled with the field-measured leaf nitrogen levels for empirical modeling of canopy nitrogen content based on first derivatives, continuum-removed reflectance and ratio-based indices in the 562–600 nm range. Results indicated that the best-performing model type varied between in situ and aircraft data in different months. However, models from the pooled samples over the growing season with acceptable accuracy suggested that these methods are robust with respect to canopy heterogeneity across spatial and temporal scales.