Surfactin is a cyclic lipopeptide produced by Bacillus spp., consisting of a β-hydroxy fatty acid and a heptapeptide synthesized by non-ribosomal peptide synthetases. Surfactin congeners (A, B, and C) differ in amino acid substitutions, with Leu7 in surfactin A replaced by Val in B and Ile in C. Our LC–MS analysis revealed that the elution profiles of surfactin-producing strains could be classified into two distinct patterns under identical culture conditions, corresponding to surfactin A and C production. This suggests that endogenous factors influence surfactin production. Therefore, we aimed to identify the genetic factor that regulates surfactin congener production. The srfA operon for surfactin A biosynthesis in B. subtilis, composed of four open reading frames (ORFs), is srfAABCD. Comparative genomic analysis between the B. subtilis JCM 1465 srfA operon and the TUA12 surfactin biosynthesis genes examined in this study revealed that the operon responsible for surfactin A biosynthesis is distinct, exhibiting 68.7%, 69.2%, 84.7%, and 67.4% homology with the four ORFs, respectively. Similarly, the operon for Ptrs2 surfactin C biosynthesis showed 68.7%, 69.2%, 64.4%, and 67.1% homology. These differences indicate that the identified surfactin A and C biosynthetic operons are novel genetic variants. Further analysis identified the adenylation domain responsible for selecting Ile7 in surfactin C via domain substitution in a surfactin A-producing strain. Average nucleotide identity analysis showed that the surfactin A and C operons were found in B. velezensis and B. amyloliquefaciens, respectively. Our findings suggest that surfactin congener production is species-dependent, with the srf operon specifically distributed in Bacillus spp.