ABSTRACT Suppressive subtractive hybridization (SSH) was used to identify genetic markers associated with biological control of plant pathogens by Bacillus subtilis. The genomes of two commercialized strains, GB03 and QST713, were compared with that of strain 168, which has no defined biocontrol capacities, to obtain a pool of DNA fragments unique to the two biocontrol strains. The sequences of 149 subtracted fragments were determined and compared with those present in GenBank, but only 80 were found to correspond to known Bacillus genes. Of these, 65 were similar to genes with a wide range of metabolic functions, including the biosynthesis of cell wall components, sporulation, and antibiotic biosynthesis. Sixteen subtracted fragments shared a high degree of similarity to sequences found in multiple B. subtilis strains with proven biocontrol capacities. Oligonucleotide primers specific to nine of these genes were developed. The targeted genes included five genes involved in antibiotic synthesis (bmyB, fenD, ituC,srfAA, and srfAB) and four additional genes (yndJ, yngG, bioA, and a hypothetical open reading frame) not previously associated with biological control. All nine markers were amplified from the commercialized B. subtilis strains GB03, QST713, and MBI600, with the exception of ituC, which was not detected in GB03. The markers also were amplified from four other B. subtilis isolates, but they were not amplified from other related Bacillus strains, including the plant growth-promoting rhizobacteria IN937a and IN937b. Sequencing of the amplified markers revealed that all seven of the isolates that scored positive for multiple markers were genotypically distinct strains. Interestingly, strains scored positive for the amplifiable markers generally were more effective at inhibiting the growth of Rhizoctonia solani and Pythium ultimum than other Bacillus isolates that lacked the markers. The potential utility of the defined genetic markers to further define the diversity, ecology, and biocontrol activities of B. subtilis are discussed.