Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease in cattle and other ruminants, and it is also a possible cause of Crohn's disease in humans. We have initiated studies to elucidate the biochemical differences that allow MAP to maintain a specific biological niche that is not shared by the very closely related Mycobacterium avium subsp. avium (MAA), an opportunistic human pathogen that causes systemic infections in immunocompromised individuals, respiratory diseases (e.g. "hot tub lung") in the general population, and unilateral lymphadenitis in young children. Here we report the comparative lipidomics of MAP vs. other members of the M. avium complex (MAC). These studies were initiated through a global lipidomic analysis of MAP strain K-10 and MAA strain 2151 (serovar 2). Total lipids of lyophilized whole cells and of lyophilized culture filtrate were extracted with chloroform/methanol (2:1) and the non-lipid components were removed by Folch wash. Lipids were separated by two-dimensional thin layer chromatography using five different solvent systems and visualized by different staining reagents to deduce the chemical nature of these lipids. Nine lipids have been identified in MAP strain K-10 that are absent from MAA strain 2151. A more comprehensive analysis with strains representing all 28 serovars of the M. avium complex demonstrated that four of the lipids were truly MAP-specific; two polar lipids, one apolar phospholipid, and one apolar lipopeptide complex. These four lipids were only associated with whole cells of MAP and did not appear to be present in the culture filtrate.