The Bacillus cereus group includes Bacillus anthracis, B. cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. The small acid soluble spore protein (SASP) beta has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP beta of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP alpha is conserved in sequence among these two species. Neither SASP alpha nor beta for B. thuringiensis, B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP alpha and beta demonstrated consistent MW among B. cereus, B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP alpha and beta were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP alpha (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP beta (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP alpha-beta type, was described here for the first time.