Biological Nitrogen Fixation is critical for ecosystem productivity. Select members of Bacteria and Archaea express a nitrogenase enzyme complex that reduces atmospheric nitrogen to ammonia. Several nitrogen fixing bacteria form symbiotic associations with plants, but free-living diazotrophs also contribute a substantial amount of nitrogen to ecosystems. The aim of this study was to isolate and characterize free-living diazotrophs in arid lands of South Dakota Badlands. Samples were obtained from sod tables and the surrounding base in spring and fall. Diazotrophs were isolated on solid nitrogen free medium (NFM) under hypoxic conditions, and their16S rRNA and nifH genes sequenced. nifH was also amplified directly from soil DNA extracts. The 16S rRNA gene data indicated a diversity of putative free-living diazotrophs across 4 phyla (Actinomycetes, Proteobacteria, Bacteroidetes, and Firmicutes), but ∼50% of these clustered with Streptomyces. These Streptomyces isolates grew in liquid NFM in an ammonia-depleted environment. Only 5 of these yielded a nifH gene product using the PolF/PolR primer set. Four of these aligned with nifH of the cyanobacteria Scytonema and Nostoc, and the other one aligned with nifH of Bradyrhizobium. Six selected Streptomyces isolates, three of which were nifH positive by PCR, all indicated N incorporation, providing strong support of nitrogen fixation. All nifH amplicons from soil DNA extract resembled Cyanobacteria. This is the first known report of diazotrophic Streptomyces, other than the thermophilic, autotrophic S. thermoautotrophicus. nifH genes of these Streptomyces were related to those from Cyanobacteria. It is possible that the cyanobacteria-like nifH amplicons obtained from soil DNA were associated with Streptomyces.