Phylogenetically diverse Ensifer strains associated with five species of Tephrosia growing in alkaline soils of semi-arid regions of the Thar Desert were characterized using multi locus sequence analysis. Based on 16S rRNA and four protein-coding housekeeping gene (recA, atpD, glnII and dnaK) sequences, the Tephrosia-Ensifer strains were genetically different from the type strains of Ensifer saheli, Ensifer kostiensis, Ensifer terangae (African origin) and Ensifer psoraleae (Asiatic origin). One strain, Ensifer sp. TL4, showed maximum similarity (99%) to Ensifer adhaerens LMG 20216(T) and formed a separate lineage close to it. Phylogenetic incongruence between sym and housekeeping genes was observed. The monophyletic origin of symbiotic gen...