Symbiotic N2 fixation capacity may affect productivity of the perennial legume Illinois bundleflower [Desmanthus illinoensis (Michx.) MacMill. ex B.L. Rob. & Fernald]. Our objective was to estimate N2 fixation of three Illinois bundleflower accessions managed for forage. Herbage yield ranged from 1.02 Mg ha-1 to 3.69 Mg ha-1 in Year 1, and 2.99 Mg ha-1 to 8.27 Mg ha-1 in Year 2. Accessions differed in herbage yield, aboveground N yield, and N2 fixed at certain locations in Year 1, but not in Year 2. Percentage of N derived from the atmosphere (%Ndfa) varied with location but not with accession in either year. The 15N natural abundance method gave lower estimates of %Ndfa than the 15N enrichment method. In yr 1, N2 fixation ranged from 0 to 30 kg ha-1 N (15N natural abundance method), 11 to 43 kg ha-1 (15N enrichment method), and 0 to 50 kg ha-1 N (total N difference method), and in Year 2 these estimates at two locations were 60 to 67 kg ha-1 N, 79 to 127 kg ha-1 N, and 67 to 142 kg ha-1 N, respectively. Differences in N2 fixation among locations could have been due to rhizobial strains. At the high-yielding location with the greatest N 2 fixation, over one-half of the nodules were occupied by indigenous rhizobial strains, whereas at other locations, strains from commercial inoculant accounted for most nodules. Symbiotic N2 fixation by Illinois bundleflower could be enhanced by new rhizobial inoculums.