Technical Abstract: Bacillus mojavensis, RRC101 is an endophytic bacterium patented for control of fungal diseases in maize and other plants. DNA fingerprint analysis of the rep-PCR fragments of 35 B. mojavensis and 4 B. subtilis strains using the Diversilab genotyping system revealed genotypic distinctive strains along geographical origins. Culture extracts from these strains, RRC 101 were antagonistic to the pathogenic and mycotoxic fungus, Fusarium verticillioides. The identity of the inhibitory substance was recently determined to be the biosurfactant, surfactin. The absolute structure of this cyclic lipopeptide was determined from collisional ion dissociation (CID) analysis to be a cyclic heptapeptide linked to a ß-hydroxy fatty acid. Further, CID analysis of the peptide moiety was established by deduction, which indicated that the peptide sequence consisted of two acidic amino acids and five hydrophobic amino acids with a sequence of Leu-Leu-Asp-Val-Leu-Leu-Glu. These spectra indicated that B. mojavensis RRC 101 produced Leu7-surfactin, and is toxic to F. verticillioides. We examined 32 additional strains of this species for production of this cyclic lipopeptide and its isomers, which were identified by HPLC-MS analysis and measured. Eighteen isolates produced several isomers of surfactin when cultured on a specific medium. This is the first report of this very powerful biosurfactant but environmental friendly biopesticide from this endophytic species.