Prairies used for bioenergy production have potential to generate marketable products while enhancing environmental quality, but little is known about how prairie species composition and nutrient management affect the suitability of prairie biomass for bioenergy production. We determined how functional-group identity and nitrogen fertilization affected feedstock characteristics and estimated bioenergy yields of prairie plants, and compared those prairie characteristics to that of corn stover. We tested our objectives with a field experiment that was set up as a 5 × 2 incomplete factorial design with C3 grasses, C4 grasses, legumes, and multi-functional-group mixtures grown with and without nitrogen fertilizer; a fertilized corn treatment was also included. We determined cell wall, hemicellulose, cellulose, and ash concentrations; ethanol conversion ratios; gross caloric ratios; aboveground biomass production; ethanol yields; and energy yields for all treatments. Prairie functional-group identity affected the biomass feedstock characteristics, whereas nitrogen fertilization did not. Functional group and fertilization had a strong effect on aboveground biomass production, which was the major predictor of ethanol and energy yields. C4 grasses, especially when fertilized, had among the most favorable bioenergy characteristics with high estimated ethanol conversion ratios and nongrain biomass production, relatively high gross caloric ratios, and low ash concentrations. The bioenergy characteristics of corn stover, from an annual C4 grass, were similar to those of the biomass of perennial C4 grasses. Both functional-group composition and nitrogen fertility management were found to be important in optimizing bioenergy production from prairies.