The soil bacterium Bacillus subtilis represents bacteria that produce a series of peptide antibiotics. They are members of both classes: the ribosomally synthesized e.g. subtilin (Gross et al., 1973; Banerjee and Hansen, 1988), Ericin (Stein et al., 2002) and sublancin (Palk et al., 1998) and the nonribosomally synthesized such as the lipopeptides surfactin (Arima et al., 1968; Leenders et al., 1999), mycosubtilin (Peypoux et al., 1976; Duitman et al., 1999), and fengycin (Umezawa et al., 1986; Vanittanakom et al., 1986; Steller et al., 1999), Bacilysocin (Tamehiro et al., 2002) and 3,3'-Neotrehalosadiamine (Inaoka et al., 2004). Subtilosin A is one of many antibiotics produced by Bacillus strains (Babasaki et al., 1985; Zheng et al., 1999; Stein et al., 2004) its importance and role in Bacillus group is little understood. Subtilosin is a macrocyclic structure (Fig. 1C) with three inter-residual linkages (Marx et al., 2001) that have been elucidated as thioether bonds between cysteine sulphurs and amino acid alpha-carbons (Kawulka et al., 2004). An acidic isoelectric point differentiates subtilosin from the basic lantibiotics (Jack and Jung, 2000; Sahl and Bierbaum, 1998). In subtilosin posttranslational linkage of a thiol to the R-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, and very rare in secondary metabolites (Kawulka et al., 2004). The mature product is formed by loss of an unusually short seven amino acid leader peptide, cyclization of the N and C termini, and further modification of Cys, Thr, and Phe residues (Zheng et al., 1999). The mature subtilosin peptide is highly resistant to enzymatic proteolysis and is stable to moderate heat and acid treatment. It acts against a variety of Grampositive bacteria, including Listeria (Zheng et al., 1999). The production of mature subtilosin is based on the expression of the sbo-alb gene cluster encompassing the subtilosin structural gene sbo and genes involved in posttranslational modification and processing of presubtilosin and in immunity (Zang et al., 1999; Zang et al., 2000). Expression of the sbo-alb genes occurs under stress conditions (Nakano et al., 2000).16S rRNA gene was used for rapid identification of the Bacillus genus was undertaken by Goto et al. (2000) and Fox et al. (1992). At this time, the validity of using a hypervariable region (nucleotides 70 to 344) of the gene was proven adequate to discriminate between all the species except between B. cereus and B. anthracis and between B. mojavensis and B. atrophaeus. The high 16S rRNA gene sequence similarities between some strains within this genus can even share phenotypic properties, however, they have been classified as different species based on DNA association values hence, demonstrated the need for a polyphasic approach to the systematics of this genus (Blackwood et al., 2004). This was observed between B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii, which share phenotypic profiles but have segregated based on DNA reassociation values of 58 to 69%, in addition to minor polymorphisms in the 16S rRNA gene between the type strains (Nakamura et al., 1999). Further, B. mojavensis and B. subtilis subsp. spizizenii have only a 1-bp difference in the 16S rRNA gene and can only be distinguished from each other by sexual isolation, divergence in DNA sequences of the rpoB and gyrA genes, and fatty acid composition (Nakamura et al., 1999). Recently an article was published (Stein et al., 2004) pre-postulate the coding gene subtilosin gene (sbo) to develop evolutionary divergence in B. subtilis subspecies too. This report is to describe subtilosin production by 16 wild-type B. subtilis strains and B. amyloliquefaciens. The sbo genes of these organisms were sequenced in order to analyze the genetic variation between B. subtilis wild-type strains. The PCR screening was correlated to production of subtilosin A.