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Background: Bacille Calmette–Guerin (BCG)-related complications can occur in vaccinated children. Comparison of the composition of cellular proteins of virulent Mycobacterium tuberculosis (MTB) H37Rv with of attenuated Mycobacterium bovis BCG Tokyo172 vaccine strain used in Thailand and identify protein candidates of value for differentiation between the two mycobacterial species may facilitate the diagnosis of etiologic agent of mycobacterial disease in vaccinated children, as most cases have been believed to have originated from BCG vaccine. Materials and Methods: The two-dimensional electrophoresis (2DE) proteomic profiles of cellular proteins from the Thai vaccine strain M. bovis BCG Tokyo172 and MTB were compared and the matched spots in 2DE gels were submitted to mass spectrometry analysis. Results: There were a number of similar protein contents with different intensity or position between MTB and M. bovis BCG Tokyo172. A higher expression of some immunogenic proteins was shown in BGG Tokyo172 when compared to MTB, while some were shown the opposite pattern. Conclusions: Proteomic approach reveals key proteins participating in different species of Mycobacteria, and may be useful for discrimination between MTB and the BCG Tokyo172 infection.