The use of synthetic nitrogen fertilizers to grow biofuel crops adds to the economic and environmental costs of biomass production. Biological nitrogen fixation provides an alternative, eco-friendly source of nitrogen for leguminous plants and some nonlegumes. With the objective of characterizing and eventually harnessing nitrogen-fixing bacteria in switchgrass roots, we assayed nitrogenase activity and surveyed nifH-expressing bacteria in roots of switchgrass grown under agricultural conditions. Plants were cultivated at two separate locations in Oklahoma in different soil types for 5 years with three nitrogen fertilizer regimes (0, 90, and 180 kg of N ha⁻¹ year⁻¹). Nitrogenase activity associated with roots cleaned of soil was found to be highest in plants grown with no N fertilizer and lowest or not detectable for plants grown at the highest level of N fertilizer. A total of 454 abundant nifH operational taxonomic units (OTUs) were identified in the switchgrass roots. Diversity analysis of active nifH-expressing bacteria showed that the most common orders were Burkholderiales (171 OTUs), Desulfovibrionales (55 OTUs), and Rhizobiales (44 OTUs). OTUs belonging to Azonexus, Pseudacidovorax, Desulfovibrio, Dechloromonas, and Bradyrhizobium were enriched in roots of plants grown under low nitrogen conditions. Analysis of significant OTUs showed distinct nifH-expressing microbial communities at the two field locations, as well as a unique community in roots of unfertilized soil at Frederick that exhibited the highest rates of nitrogen fixation. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .