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At two sites in the North Central USA (Michigan (KBS) and Wisconsin (ARL)), we evaluated the effect of N fertilization on the yield and quality of five perennial bioenergy feedstock cropping systems: (1) switchgrass (Panicum virgatum L.), (2) giant miscanthus (Miscanthus × giganteus), (3) a native grass mixture (5 species), (4) an early successional field (volunteer herbaceous species), and (5) a restored prairie (18 species). In a randomized complete block design with 5 replicates and 2 split plots, N was applied at 0 and 56 kg ha−1 to split plots for each cropping system from 2010 to 2016. No yield response to N was detected in switchgrass at either location in any year. Giant miscanthus exhibited a positive yield response to N at both sites (11% at KBS and 83% at ARL). Nitrogen fertilizer addition significantly reduced glucose (KBS 12.9 and 13.8 g kg−1 year−1, ARL 11.2 and 9.7 g kg−1 year−1) in the native grass mix and restored prairie systems respectively. Nitrogen fertilizer also reduced xylose at KBS in the switchgrasss, native grass mix, and restored prairie (4.9, 7.5, and 5.0 g kg−1 year−1). At ARL, N fertilization reduced xylose levels in switchgrass, giant miscanthus, and restored prairie (7.4, 6.8, and 6.2 g kg−1 year−1) and increased xylose levels in the early successional system (5.0 g kg−1 year−1).