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Francisella tularensis, the causative agent of tularaemia, is a potential agent of bioterrorism. The phenotypic discrimination of the closely related F. tularensis subspecies and individual strains with traditional methods is difficult and time consuming, often producing ambiguous results. Surface-enhanced laser desorption/ionization time-of-flight MS (SELDI-TOF MS) was used in this study to discriminate the different species and subspecies of the genus Francisella.