Perennial grasses can assimilate nitrogen (N) fixed by non-nodulating bacteria living in the rhizosphere and the plant's own tissues, but many details of associative N fixation (ANF) remain unknown, including ANF's contribution to grass N nutrition, the exact location of fixation, and composition of the associated microbial community. We examined ANF in switchgrass (Panicum virgatum L.), a North American perennial grass, using 15N-enriched N2 isotopic tracer additions in a combination of in vitro, greenhouse, and field experiments to estimate how much N is assimilated, where fixation takes place, and the likely N-fixing taxa present. Using in vitro incubations, we documented fixation in root-free rhizosphere soil and on root surfaces, with average rates of 3.8 μg N g root−1 d−1 on roots and 0.81 μg N g soil−1 d−1 in soil. In greenhouse transplants, N fixation occurred only in the early growing season, but in the field, fixation was irregularly detectable throughout the 3-month growing season. Soil, leaves, stems, and roots all contained diazotrophs and incorporated fixed N2. Metagenomic analysis suggested that microbial communities were distinct among tissue types and influenced by N fertilizer application. A diverse array of microbes inhabiting the rhizosphere, and possibly aboveground tissues, appear to be episodically contributing fixed N to switchgrass.