Aims: To rapidly type the fengycin homologues produced by Bacillus subtilis strains with electrospray ionization/ collision-induced dissociation (ESI/CID) mass spectrometry. Methods and Results: Fengycin homologues produced by Bacillus subtilis JA were analysed. When each homologue was subjected to ESI/CID analysis, ions representing characteristic fragmentations were detected. These ions can help to identify the homologues; even homologues of the same nominal mass can be discriminated by their ESI/CID spectra. Based on the CID results, fengycin homologues can be correctly assigned. Conclusions and Significance of this Study: ESI/CID leads to rapid detection and structural characterization of fengycin homologues or lipopeptides with similar properties. It will be very useful in studying the regulatory expression of these peptides.