Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied for the characterization of Bacillus anthracis spore biomarkers. B. anthracis spores were extracted under a simple procedure, followed by linear mode analysis, using sinapinic acid as the matrix. Several markers with a mass range of 4-7 kDa were detected in three B. anthracis strains: Vollum, Sterne and V770-NP1-R. Similar spectra were also obtained for spore extracts of two members of the B. cereus group: B. thuringiensis and B. cereus, but not for B. mycoides, B. subtilis or B. licheniformis, suggesting that these markers are specific to closely related members of the B. cereus group. When alpha-cyano-4-hydroxycinnamic acid was used as the matrix, at least four additional new markers within a mass range of 2-4 kDa could be detected in all B. anthracis spore extracts. These markers, corresponding to a molecular weight of 2528.3, 2792.4, 3077.4, and 3590.7 Da, have not been observed in extracts of the three closely related Bacillus species - B. cereus, B. thuringiensis and B. mycoides. These unique B. anthracis biomarkers, which were isotopically resolved and reproducibly detected in the highly accurate MALDI-TOFMS reflectron mode, may be useful as a basis for rapid and specific identification of B. anthracis strains.