Biological N2 fixation (BNF) via legume intercrops can provide an alternative to inorganic N fertilizer and thereby minimize the economic and environmental costs of bioenergy feedstock and forage production. The objectives of this study were to (i) verify switchgrass (Panicum virgatum L.) as a non-N2-fixing reference plant for distinguishing between soil- and atmosphere-derived N; (ii) determine BNF levels via the15N enrichment method for one cool-season legume (red clover [Trifolium pratense L.]) and three warm-season legumes ([partridge pea [Chamaecrista fasciculate Michx.], sunn hemp [Crotalaria juncea L.], and pigeonpea [Cajanus cajan (L.) Millsp.]) when intercropped into switchgrass and a near relative, guineagrass (Panicum maximum L.); and (iii) evaluate the validity of the N-difference method by comparing it against the15N enrichment technique in humid temperate and semiarid tropical climates. The results revealed little difference in the N assimilation rates of legume and non- N2-fixing reference plants, suggesting that switchgrass is an appropriate reference species. Annual fixation for red clover was greatest, followed by partridge pea, and lastly sunn hemp in temperate systems (87, 84, and 35 kg ha-1, respectively), all of which tended to supply greater amounts of N in subsequent seasons. Considerably greater BNF occurred in tropical intercrop systems (exceeding 240 kg ha-1). Consequently, the BNF of selected legumes approximates or exceeds recommended N fertilizer rates (67 kg N ha-1) in both humid temperate and semiarid tropical pasture and feedstock systems. The N-difference method did not provide estimates for BNF that were comparable to 15N-enrichment-derived values (P > 0.05).