Aims: To determine the composition of polar glycopeptidolipids (pGPLs) of Mycobacterium simiae and, particularly, those of 'habana' strains, in a search for specific markers given the immunogenic potential of 'habana' TMC 5135 in experimental tuberculosis. Methods and Results: pGPLs were determined in free lipid extracts using electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS), working in both negative- and positive-ion mode. In the case of TMC 5135, the presence of the previously characterized GPL-II (containing 2,4-di-O-CH3 glucuronic acid as distal sugar in the oligosaccharide antigenic moiety) and GPL-III (containing 4-O-CH3 glucuronic acid as distal sugar) was confirmed using MS/MS and MS/MS/MS approaches. Interestingly, some 'habana' strains presented variants of GPL-II, designated GPL-II'-A and GPL-II'-B. A di-O-CH3-deoxy-hexose (tentatively, 2,3-di-O-CH3-fucose) was identified as the penultimate sugar in the oligosaccharide moiety of GPL-II'-A, whereas in GPL-II'-B the penultimate sugar was fucose (tentative identification). On the contrary, the distal sugar of the oligosaccharide chain of pGPLs of Myco. simiae ATCC 25275(T) was identified as tri-O-CH3-glucuronic acid (designated GPL-sim(T)-I, with two variants: GPL-sim(T)-I-A and GPL-sim(T)-I-B), O-CH3-glucuronic acid (designated GPL-sim(T)-II) and di-O-CH3-glucuronic acid (GPL-II'-A and GPL-II'-B). The penultimate sugar of the oligosaccharide chain of GPL-sim(T)-I-A and GPL-sim(T)-II was identified as di-O-CH3-deoxy-hexose (tentatively, 2,3-di-O-CH3 fucose), and that of GPL-sim(T)-I-B as deoxy-hexose (tentatively, fucose). In all strains studied, each [M-H](-) and [M+Na](+) ion was revealed as a mixture of homologous compounds varying in the number of -O-CH3 groups present in the oligosaccharide moiety and in the length of the fatty acyl linked to the peptide. Conclusions: The present work indicates that, within a similar general pattern of pGPLs, different strains of Myco. simiae present some variations, so that new compounds (GPL-II'-A, GPL-II'-B, GPL-sim(T)-I-A, GPL-sim(T)-I-B and GPL-sim(T)-II) were defined. Noteworthy was the fact that the 'habana' strains clearly differed from the type strain of Myco. simiae. Significance and Impact of the Study: The data obtained can be used in the delineation of the 'habana' group of Myco. simiae, including the quality control of the immunogenic strain 'habana' TMC 5135.