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The tree Senegalia (Acacia) senegal is well adapted to soils of arid regions having a vital role in reforestation and increasing soil fertility through biological nitrogen fixation in addition to its utility as edible seeds and gum. Fifty-nine root nodule bacterial (RNB) strains were isolated; characterized at phenotypic and genotypic level; and host range was studied. The Senegalia-RNB strains were able to grow up to 3% NaCl and on a wide range of pH (5-11) with optimum growth at alkaline pH showing their adaptation to alkaline desert soil. The carbon utilization pattern of Senegalia-RNB strains was significantly different from existing closely related type strains of Ensifer. The PCR-RFLP (ARDRA) as well as RAPD patterns revealed considerable genetic diversity among the strains forming distinct genetic groups. The 16S rRNA phylogeny of selected six (AS11, AS18, AS27, AS34, AS50 and AS53) strains showed that they belong to fast growing species of Ensifer with few being similar to the previously described Ensifer strains from Vachellia jacquemontii and Vachellia leucophloea from Thar Desert of India. The symbiotic (nifH and nodA) gene phylogeny showed incongruence with that of 16S rRNA gene (species) phylogeny suggesting HGT incidences under hot-arid and alkaline environment. The Senegalia-Ensifer strains possessing novel symbiotic genes could effectively cross-nodulate species of Vachellia (V. jacquemontii, V. leucophloea and V. nilotica) but failed to nodulate the crop Vigna radiata. Multi locus sequence analysis (MLSA) is required to determine novelty of these Ensifer strains and more cross-inoculation studies will help in understanding their potential use as inoculums in nursery practices for reforestation program in arid and semi-arid regions.