Mycobacterium tuberculosis (Mtb) lipids including glycolipids and lipoglycans play a crucial role in the modulation of the host immune response by targeting the innate receptors C-type lectins, TLRs and the CD1 proteins of class 1. Glycolipids have been shown to be biomarkers of M. tuberculosis strains and also of opportunistic mycobacteria called non-tuberculous mycobacteria. Most of the structural and functional work of the Mtb lipids has been done using lipids arising from M. tuberculosis cell growth in vitro. However it is likely that lipid structures can change during infection or among the M. tuberculosis or opportunistic clinical strains. Here we describe a new, rapid and sensitive analysis of lipids directly on whole mycobacteria which can be done in few minutes and on less than 1000 mycobacteria by direct matrix-assisted laser desorption/ionization mass spectrometry using an unusual solvent matrix. By this new methodology, which does not require extraction or purification steps, we are able to discriminate mycobacteria belonging to the Mtb complex as well as opportunistic and non-pathogenic mycobacteria. This method was also found to be successful for identification of an envelope lipid mutant. This work opens a new analytical route for in vivo analysis of mycobacterial lipids.