Two types of colonies with different morphologies (smooth: S and rough: R) formed when Bacillus Calmette-Guérin (BCG) Tokyo172 substrain was cultured on Middlebrook 7H10 agar medium, and their genotypes were analyzed by multiplex PCR on five RD regions and SenX3-RegX3. In most cases these two colony types had different genotypes, i.e., S colonies showed a characteristic 22 bp deletion in Rv3405c of the RD16 region (type I), and R colonies did not have this deletion (type II) similar to many other BCG substrains. Thus, there was a strong relationship between colony morphology and genotype. Both genotypes were found in every Tokyo172 preparation tested, including the seed lot for production, the origin of seed lot from the 1960s and ATCC BCG Japan. Type I was always in the majority. It was suggested that types I and II constituted independent subpopulations within the Tokyo172 substrain. Type I was shown to have a growth advantage over type II both on culture media and in mice organs.