Mycobacterium tuberculosis, the causative agent of tuberculosis, is unique among bacterial pathogens in that it contains a wide array of complex lipids and lipoglycans on its cell wall. Among them, the sulfated glycolipid, termed the sulfolipid, is thought to mediate specific host-pathogen interactions during infection. Sulfolipids (SLs), including sulfolipid I (SL-I) and sulfolipid II (SL-II), are 2,3,6,6′-tetraacyltrehalose 2′-sulfates. SL-I was identified as a family of homologous 2-palmitoyl(stearoyl)-3-phthioceranoyl-6, 6′-bis(hydroxyphthioceranoy1)trehalose 2′-sulfates and was believed to be the principal sulfolipid of M. tuberculosis strain H37Rv. We cultured and extracted sulfolipids using various conditions, including those originally described, and employed high-resolution multiple-stage linear ion-trap mass spectrometry with electrospray ionization to characterize the structure of the principal SL. We revealed that SL-II, a family of homologous 2-stearoyl(palmitoyl)-3,6,6′-tris(hydroxyphthioceranoy1)trehalose 2′-sulfates, rather than SL-I is the principal sulfolipid class. We identified a great number of isomers resulting from permutation of the various hydroxyphthioceranoyl substituents at positions 6 and 6′ of the trehalose backbone for each of the SL-II species in the entire family. We redefined the structure of this important lipid family that was misassigned using the traditional methods 40 years ago.