This study evaluates the carbon fluxes and trends and examines the environmental sustainability (e.g., carbon budget, source or sink) of the potential biofuel feedstock sites identified in the Greater Platte River Basin (GPRB). A 9-year (2000–2008) time series of net ecosystem production (NEP), a measure of net carbon absorption or emission by ecosystems, was used to assess the historical trends and budgets of carbon flux for grasslands in the GPRB. The spatially averaged annual NEP (ANEP) for grassland areas that are possibly suitable for biofuel expansion (productive grasslands) was 71–169 g C m−2 year−1 during 2000–2008, indicating a carbon sink (more carbon is absorbed than released) in these areas. The spatially averaged ANEP for areas not suitable for biofuel feedstock development (less productive or degraded grasslands) was −47 to 69 g C m−2 year−1 during 2000–2008, showing a weak carbon source or a weak carbon sink (carbon emitted is nearly equal to carbon absorbed). The 9-year pre-harvest cumulative ANEP was 1166 g C m−2 for the suitable areas (a strong carbon sink) and 200 g C m−2 for the non-suitable areas (a weak carbon sink). Results demonstrate and confirm that our method of dynamic modeling of ecosystem performance can successfully identify areas desirable and sustainable for future biofuel feedstock development. This study provides useful information for land managers and decision makers to make optimal land use decisions regarding biofuel feedstock development and sustainability.