Diversity among biosurfactant producing Bacillus spp. from diverse habitats was studied among 77 isolates. Cluster analysis based on phenotypic characteristics using unweighted pair-group method with arithmetic averages (UPGMAs) method was performed. Bacillus isolates possessing high surface tension activity and five reference strains were subjected to amplified 16S rDNA restriction analysis (ARDRA). A correlation between the phenotypic and genotypic characterization of Bacillus spp. is explored. Most of the oil reservoir isolates showing high surface activity clustered with B. licheniformis and B. subtilis, the hot water spring isolates clustered in two ingroups, while the petroleum contaminated soil isolates were randomly distributed in all the three ingroups. Present work revealed that diversity exists in distribution of Bacillus spp. from thermal and hydrocarbon containing habitats where majority of organisms belonged to B. licheniformis and B. subtilis group. Isolate B. licheniformis TT42 produced biosurfactant which reduced the surface tension of water from 72 mNm(-1) to 28 mNm(-1), and 0.05 mNm(-1) interfacial tension against crude oil at 80°C. This isolate clustered with B. subtilis and B. licheniformis group on the basis of ARDRA. These findings increase the possibility of exploiting the Bacillus spp. from different habitats and their possible use in oil recovery.