Associative nitrogen fixing (ANF) microorganisms in feedstock grasses enhance the soil nitrogen pool in order to supply nitrogen required under natural field conditions. Perennial feedstock grass can yield large quantities of lignocellulose useful for the production of biofuels. Such grasses include Miscanthus x giganteus (Mxg), Pancium virgatum (switchgrass), and energycane (US 02-147). Endophytic and free-living associative nitrogen fixing bacteria often support these grasses to meet the required nitrogen needs of the plants. The objective of this study was to measure the potential for associative nitrogen fixation in these three feedstock grasses over a three-year field study to determine their ability to accumulate nitrogen in the soil. Total above ground biomass, δ ¹⁵N in soil collected at two depths (0-10 and 10-30 cm), and δ ¹⁵N in roots and shoots were analyzed to determine the collective nitrogen contribution to the soil for each grass. The soil δ ¹⁵N values collected at the 0-10 cm depth significantly decreased in energycane in comparison to switchgrass and giant miscanthus. Moreover, the δ ¹⁵N in roots of energycane were reduced drastically, suggesting endophytic nitrogen fixating microorganisms contribute to the increased plant nitrogen pool. Reductions were also evident with switchgrass and giant miscanthus roots. All three energy grasses had lower δ ¹⁵N than the annual Sorghum (M81-E) and soil alone. These results indicate that energycane associated nitrogen fixers have the highest potential to fix nitrogen in soil under natural field condition.