In order to evaluate the biochemical characteristics of 14 substrains of Mycobacterium bovis bacillus Calmette Guérin (BCG) – Russia, Moreau, Japan, Sweden, Birkhaug, Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia and Pasteur – we performed eight different biochemical tests, including those for nitrate reduction, catalase, niacin accumulation, urease, Tween 80 hydrolysis, pyrazinamidase, p-amino salicylate degradation and resistance to thiophene 2-carboxylic acid hydrazide. Catalase activities of the substrains were all low. Data for nitrate reduction, niacin accumulation, Tween 80 hydrolysis, susceptibility to hydrogen peroxide and nitrate, and optimal pH for growth were all variable among these substrains. These findings suggest that the heterogeneities of biochemical characteristics are relevant to the differences in resistance of BCG substrains to environmental stress. The study also contributes to the re-evaluation of BCG substrains for use as vaccines.