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Bacillus spp. is a potential bacterial antagonist to manage bacterial wilt disease of tomato incited by Ralstonia solanacearum, which is one of the most threatening diseases of tomato in India. Genetic diversity of Bacillus strains and their potentiality to control bacterial wilt of tomato isolated from rhizospheric soil and endophytic tomato plants from different agro-climatic regions of India were studied. Rhizospheric soil and plants of tomato were pasteurized at 80°C for 15 min before dilution and then inoculated onto the Petri plates containing tryptic soy agar medium and incubated at 28± 2°C. Out of 250 isolates of Bacillus species, 47 strains showed antagonistic ability against R. solanacearum. Maximum growth of R. solanacearum was inhibited by strain DTBS-5 to form inhibition zone of 5.5 cm2 in vitro and lowest wilt incidence of 14.3 and 7.6 % in Pusa Ruby and Arka Abha cultivars under glass house conditions, respectively. Plants treated with strain JTBS-9 had maximum fresh weight of 42.0 and 49.0 g and dry weight 6.1 and 6.6 g in tomato cultivars Pusa Ruby and Arka Abha after 45 days of transplanting, respectively. Out of 47 strains, 11 strains of Bacillus spp. were detected as iturin antibiotic producing strains by using iturin gene based marker. Genetic variability was found in Bacillus spp., which was made 5 clusters at 50% similarity coefficient. However, iturin producing and iturin non-producing strains as well as rhizospheric and endophytic Bacillus spp. could not be distinguished by using 16S rRNA sequence analysis and genetic fingerprinting. Key words: BOX-PCR, Bacillus spp., iturin, Ralstonia solanacearum, phylogenetic analysis, tomato.