Aims: The aim of this work was to compare the chemical structure of the spore cortex of a range of species, and to determine any correlation between cortex structure and spore resistance properties. Methods and Results: The fine chemical structure of the cortex of Bacillus subtilis, Bacillus megaterium, Bacillus cereus and Clostridium botulinum was examined by muropeptide analysis using reverse phase HPLC. There is a conserved basic structure between peptidoglycan of these species, with the only difference being the level of de-N-acetylation of an amino sugar. In order to determine if an alteration in cortex structure correlates with heat resistance properties, the peptidoglycan structure and properties of B. subtilis spores prepared under different conditions were compared. Peptidoglycan from spores prepared in Nutrient Broth (NB) showed reduction in single L-alanine substituted muramic acid to only 13.9% compared with 20.6% in CCY-grown spores. NB-prepared spores are also unstable, with 161-fold less heat resistance (60 min, 85 degreesC) and 43 times less Mn2+ content than CCY-grown spores. Addition of MnCl2 to NB led to a peptidoglycan profile similar to CCY-grown spores, sevenfold more heat resistance (60 min, 85 degreesC) and an 86-fold increase in Mn2+ content. Addition of CCY salts to NB led all parameters to be comparable with CCY-grown spore levels. Conclusions: It has been shown that peptidoglycan structure is conserved in four spore-forming bacteria. Also, spore heat resistance is multifactorial and cannot be accounted for by any single parameter. Significance and Impact of the Study: Endospores made by diverse species most likely have common mechanisms of heat resistance. However, the molecular basis for their resistance remains elusive.