Biological nitrogen fixation is a major contributor of nitrogen (N) to certified organic farming systems in the United States, with presence of effective rhizobia strains essential for optimal nodulation and biological nitrogen fixation (BNF). Organic farmers have particular interest in the winter annual cover crop species hairy vetch (HV; Vicia villosa Roth). V. villosa has been shown to have high biomass production and BNF capacity, with many varieties producing over 200 kg N ha^-1 and more than 170 kg N ha^-1 of that N from BNF, however efficient inoculant strains for the Southeastern U.S. have not been developed. Our objectives were to characterize rhizobia isolated from field inoculated V. villosa root nodules and from un-inoculated adjacent soils to determine naturalized population diversity and inoculation effectiveness. Four varieties of hairy vetch were planted in three sites across North Carolina and inoculated with commercial inoculants, then rhizobia isolated from nodules of the field-grown plants. Inoculant strains were trapped from plants inoculated with commercial inoculants in sterile growth pouches, and native strains from un-inoculated adjacent soils. Strains were evaluated using BOX-PCR and sequencing to determine genetic diversity of isolated strains, followed by growth chamber evaluation of a subset of 26 genetically-distinct accessions. Growth chamber experimental units including one strain per pot were arranged in a randomized complete block design, using ten replicates of uninoculated, nitrogen-free negative controls and 4 positive fertilized controls. Genomic fingerprints indicated low levels of relatedness of inoculated field-grown plant rhizobia to recommended V. villosa inoculant strains. Sequencing of symbiotic nodC genes from isolates representative of BOX-PCR clusters showed a predominance of Rhizobia leguminosarum in native-isolated strains whereas inoculant-isolated strains were phylogenetically related to R. pisi. Growth chamber results show plants inoculated with native strains to have greater nodule number and mass than those inoculated with strains isolated from field-grown inoculated plants, but lower nitrogen concentrations.