In the present study, the potential of Fourier-transform infrared microspectroscopy to differentiate and discriminate Bacillus spp. spores that sporulated in two nonselective media were evaluated. Spore crops of Bacillus subtilis (ATCC 11779), Bacillus thuringiensis (ATCC 13368, ATCC 35866 and ATCC 55177), Bacillus amyloliquefacies (ATCC 496763) and Bacillus stearothermophilus (ATCC 7953) were obtained with Nutrient and Trypticase Soy Agar supplemented with 10ppm of MnSO4.H2O. Spores were sonicated, washed with deionized water, and pellets (108cfu/mL) were deposited onto the grids of hydrophobic membrane filters and dried to produce a uniform and thin film. Spectra were collected in the attenuated total reflectance mode in the mid-infrared region (4,000-700cm-1). Our classification models, soft independent modeling of class analogy obtained from derivatized infrared spectra (1,800-900cm-1), showed that the discrimination among spore crops was mainly attributed to infrared frequencies of spore coat proteins. © 2011 Wiley Periodicals, Inc..