Mycobacterium tuberculosis (M.tb), infects one‐third of the global population and kills two million people per year. A number of unique lipid metabolites produced by M.tb have been implicated in the virulence of the bacterium, however a comprehensive lipidomic study of differing M.tb lineages is lacking. In this study, we have sought to characterize the cell‐surface lipid profiles of M.tb laboratory strains, the virulent W/Beijing lineage, and the vaccine strain M. bovis BCG, starting with the anti‐virulence lipid S881, a sulfated menaquinone. Cell surface lipids of these strains were extracted and subject to high‐resolution mass spectrometry analysis, and the identities of the virulence lipids were confirmed via exact mass measurements with an error of < 2 ppm. Our results indicate that S881 is present in the H37Rv and CDC1551 laboratory strains, but absent in the related Erdman strain. The W/Beijing strains T85 and HN878 showed qualitatively similar levels of S881 to H37Rv, indicating that other virulence factors may play a larger role in the virulence of these strains. Finally, S881 was found to be present at qualitatively high levels in the M. bovis BCG vaccine strain, consistent with the hypothesis S881 negatively regulates the virulence of the cell. Further studies are currently underway to apply this method to the analysis of other M.tb virulence lipids. This work was supported by NIH # AI51622.