Establishment of legume-rhizobia symbiosis has ample agronomic and ecological significance. Characterization of native rhizobia could enhance our understanding of their natural distribution and co-evolution. The Great Indian Thar Desert is an ecologically significant unique habitat with its flora and fauna. Crotalaria spp. is an economically important legume widely distributed in the Thar Desert and can be considered its one of the bioresources, particularly for biological nitrogen fixation with their symbiotic rhizobia. Here, we examined the legume Crotalaria burhia Buch.-Ham. ex Benth. in search of potential novel rhizobial species. Out of 72 root nodule bacterial (RNB) strains isolated from C. burhia, 51 rhizobia-like strains were examined for genetic diversity based on ARDRA and RAPD patterns. BLASTn sequence similarity results based on 16S rRNA gene of selective thirteen strains representing four ARDRA types revealed that they were related to genera Ensifer, Rhizobium and Bradyrhizobium. In 16S rRNA gene phylogeny, five (CB5, CB17, CB36, CB44, CB56) strains were closer to Ensifer kostiensis, three (CB6, CB12, CB32) to E. terangae and CB11 showed similarity with E. kostiensis and E. saheli. Strain CB4 was similar to Bradyrhizobium yuanmingense and three (CB29, CB31, CB46) strains were closer to species of Rhizobium (R. etli, R. sullae and R. borbori respectively). Symbiotic (nodA and nifH) genes phylogeny of Ensifer sp. CB56 was incongruent and showed close similarity with E. fredii whereas sym gene phylogeny of Bradyrhizobium sp. CB4 was congruent with 16S rRNA gene phylogeny. In Rhizobium strains sym genes could not be amplified and they failed to nodulate host. Our study suggests that C.burhia is nodulated by diverse strains of Ensifer and Bradyrhizobium in alkaline soil of Thar Desert and these strains effectively cross-nodulated crop Vigna radiata.