Switchgrass (Panicum virgatum L.) is a potential biofuel crop in the midwestern United States. The objective of this experiment was to test the effect of nitrogen application on biomass dry matter yield and fiber and mineral concentrations in large field plots in Lucas and Wayne counties in southern Iowa. Two established switchgrass fields with a previous history of limited management were evaluated from 1998 through 2002. Nitrogen was applied in the spring at rates of 0, 56, 112, and 224 kg N ha−1, and a single biomass harvest was made in autumn. Biomass production averaged across locations and N levels increased by 3.6 mg ha−1 between 1998 and 2002 to 6.5 mg ha−1. Nitrogen improved yields, with the response declining as N levels increased. The highest yield throughout the experiment was 8.5 mg ha−1 at the Lucas location in 2002. Changes in fiber and mineral concentrations did not follow any trend over years but were likely due to differences in harvest date among years. Nitrogen fertilization had no meaningful effect on the quality of the biofuel produced. This study clearly shows that nitrogen application and proper agronomic management can substantially increase the yield of established switchgrass fields over time without affecting the quality of the feedstock. As this experiment was conducted in large plots using commercial farm machinery, the results should be broadly applicable to real world situations.