Information about the interaction of harvesting and N fertilization on composition of warm-season grasses grown as biofuel feedstocks is limited. Our objective was to determine composition of warm-season grasses as influenced by N fertilization rates and harvest dates. A field study was conducted near Ames, IA, during 2006 and 2007. The experimental design was a split-split plot arranged in a randomized complete block with four replications. Big bluestem (Andropogon gerardii Vitman), eastern gamagrass (Tripsacum dactyloides L.), indiangrass (Sorghastrum nutrans L. Nash), and switchgrass (Panicum virgatum L.) were main plots. Three N application rates (0, 65, and 140 kg ha(-1)) were subplots, and 10 harvest dates were sub-subplots. In both years, delaying harvest increased cellulose, lignin, and C concentrations, but decreased ash and N concentrations, however, the magnitude of these effects varied among species. On average, big bluestem had the highest cellulose (430.5 g kg(-1)) and C concentration (450.5 g kg(-1)) while switchgrass had the highest lignin concentration (58.5 g kg(-1)) among four species. Big bluestem had the lowest ash concentration (47.5 g kg(-1)) whereas indiangrass had the lowest N concentration (5.5 g kg(-1)). With increasing rates of N, average N, C, cellulose, and lignin concentrations increased 19 to 29%, 0.6 to 2.2%, 0.7 to 5.7%, and 5.6 to 23.9%, respectively, whereas ash concentration declined 1.8 to 18.4%, varied among species. Our results indicated that in general, warm-season grasses supplied with N fertilizer at 140 kg ha(-1) and harvest delayed until fall have the optimal composition for biomass feedstock production.