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Background: Molecular identification of a wide range of organisms capable of carrying out biological nitrogen fixation (BNF) are diverse in nature and significantly improves plant growth. Biological N2 fixation reflects the activity of a phylogenetically diverse list of microorganisms. Molecular characterization provides efficient means to identify organisms with the potential of N2 fixation. Applying these techniques in an array of environments has considerably broadened our understanding of the suite of organisms that can carry out BNF. Methods: Thirty-four strains of free living N2 fixing bacterial strains were isolated from diverse plants cultivated in North Gujarat, including wheat, cotton, castor and pearl millet, using a nitrogen-free selective medium. Acetylene reduction assay was used to check the ability of all bacteria to fix nitrogen. Hybridization with nifH probe derived from Azotobacter vinelandii with isolated free-living nitrogen-fixing bacteria showed a positive result. The selected strains were characterized by molecular analysis like; ARDRA and 16S rDNA sequencing. Result: Based on molecular characterization 17 strains to known groups of nitrogen-fixing bacteria, including organisms from the genus Azotobacter, Pseudomonas, Enterobacter, Arthrobacter, Bacillus, Variovorax, Nocardiodies, Rhodococcus, Mycobacterium, Planococcus, Microbacterium have been identified. One of the strains was identified as unknown bacteria. The potential strains were identified by 16srDNA analysis and also corroborated by morphological and biochemical characterization.