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Lipids are essential biomolecules that play central roles in membrane structure, energy storage, and cellular signaling. During the past two decades, lipidomics has developed into a powerful analytical approach for systematic characterization of lipid composition, metabolism, and function across biological systems. Although early lipidomic studies focused primarily on animals and vascular plants, increasing attention has been directed toward microorganisms, whose lipid diversity reflects exceptional physiological and ecological adaptability across evolutionary timescales. This review focuses specifically on the comparative structural diversity of lipids across the main microbial groups – archaea, bacteria, yeasts, fungi, cyanobacteria, algae, and viruses—and on how mass spectrometry-based lipidomics has revealed adaptive strategies to environmental stress, temperature, salinity, pH, and availability of nutrients. Microorganisms exhibit pronounced variability in lipid structures and biosynthetic pathways. Archaeal membranes are characterized by ether-linked isoprenoid lipids that provide stability under extreme environmental conditions. In contrast, bacterial membranes contain diverse phospholipids, glycolipids, and hopanoids that enable rapid membrane remodeling in response to environmental stress. Fungi and algae synthesize characteristic sterols, glycolipids, and polyunsaturated fatty acids that are essential for membrane function, cellular signaling, photoprotection, and adaptation to fluctuating growth conditions. Recent advances in high-resolution mass spectrometry, particularly liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, and ion mobility spectrometry, have enabled detailed profiling of complex microbial lipidomes and their dynamic responses to physiological and environmental stimuli. Integration of lipidomics with transcriptomic, proteomic, and metabolomic data further enhances understanding of lipid biosynthesis and regulation. This review demonstrates that microbial lipidomics provides valuable insights into microbial physiology and adaptation and supports applications in chemotaxonomy, and environmental microbiology.