Associative N fixation (ANF), the process by which dinitrogen gas is converted to ammonia by bacteria in casual association with plants, has not been well-studied in temperate ecosystems. We examined the ANF potential of switchgrass (Panicum virgatum L.), a North American prairie grass whose productivity is often unresponsive to N fertilizer addition, via separate short-term 15N2 incubations of rhizosphere soils and excised roots four times during the growing season. Measurements occurred along N fertilization gradients at two sites with contrasting soil fertility (Wisconsin, USA Mollisols and Michigan, USA Alfisols). In general, we found that ANF potentials declined with long-term N addition, corresponding with increased soil N availability. Although we hypothesized that ANF potential would track plant N demand through the growing season, the highest root fixation rates occurred after plants senesced, suggesting that root diazotrophs exploit carbon (C) released during senescence, as C is translocated from aboveground tissues to roots for wintertime storage. Measured ANF potentials, coupled with mass balance calculations, suggest that ANF appears to be an important source of N to unfertilized switchgrass, and, by extension, to temperate grasslands in general.