In this work, we applied high-resolution atomic force microscopy (AFM) to identify and characterize similarities and differences in the spore surface morphology of strains from four species of Bacilli: B. anthracis, B. cereus, B. pumilis, and B. subtilis. Common features of the examined spores in the dry state included ridges that spanned the long axis of each spore, and nanometer-scale fine rodlets that covered the entire spore surface. However, important differences in these features between species permitted them to be distinguished by AFM. Specifically, each species possessed significant variation in ridge architecture, and the rodlet width in B. anthracis was significantly less than that of the other species. To characterize similarities and differences within a species, we examined three B. subtilis strains. The ridge patterns among the three strains were largely the same; however, we detected significant differences in the ridge dimensions. Taken together, these experiments provide important information about natural variation in spore surface morphology, define structural features that can serve as species- and strain-specific signatures, and give insight into the dynamics of spore coat flexibility and its role during spore dormancy and germination.