Rhizobia diversity is considered as one of the most useful resources for bioprospecting due to their symbiotic nitrogen-fixing ability with members of Leguminosae. The highly conserved nature of the nitrogenase reductase gene (nifH) makes it an ideal molecular tool to determine the potential for biological nitrogen fixation in any environment. In the present investigation, 250 rhizobial strains were isolated from legumes belonging to different geographical locations of Chhattisgarh, India. Genetic diversity of the nitrogen-fixing bacterial community was analyzed using the nifH gene-specific primer. The polymorphism was found among the nitrogen-fixing population of different sources and origin but not in same source of rhizobia. Further, the symbiotic plasmid DNA was characterized on the basis of size and copy number of plasmids. The plasmid number varying from one to three in different rhizobial isolates had a size greater than 23 kb, while in some rhizobial isolates plasmids were absent. In addition, to examine the role of ascorbate in respiratory protection, the clear black spot margin of ascorbate was observed in the endodermis region of the nodule whereas scarcely dispersed in the infected region. Therefore, our findings demonstrated that knowing the rhizobial nifH gene diversity along with copy number of the plasmid is important for strain identification, deciding its fertility, productivity standards, and potential of biological nitrogen fixation across the geographical region.