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The surface morphology of Bacillus spores was resolved by atomic force microscopy in order to determine if characteristic surface features could be used to distinguish between closely related species. Four strains were studied: Bacillus anthracis Sterne strain, Bacillus thuringiensis var. kurstaki, Bacillus cereus strain 569, and Bacillus globigii var. niger. The spores were separated from a nutrient agar culture by filtering and centrifugation, suspended in deionized water, and immobilized on a graphite substrate by spin- coating. Atomic force microscopy was done in intermittent contact mode, in air, under reduced humidity. Height images showed that the spores had an irregular topography of subtle grooves, bumps, and steps across their upper surface. Phase images showed a superficial grain structure. Although some similarities were observed among spores of the same species, there was also significant variability within each species. The four species were not distinguished by observed surface morphology. Surface texture analysis (roughness, power spectral density, and bearing) did not establish quantitative differences between species. Overall, atomic force microscopy revealed a spore surface morphology rich with information. Additionally, it appears, that further analysis and a larger sample size could provide sufficient information to allow identification and differentiation between B. anthracis and its close relatives.