Thermophiles constitute a class of microorganisms able to grow at extremely elevated temperatures. Some of these species are classified as Gram-negative bacteria, because of the presence of an outer membrane in the cell envelope, which is located on the top of a thick murein layer. Unlike typical Gram-negative bacteria, the outer membranes of Thermus species are not composed of lipopolysaccharides but of peculiar glycolipids (GL), whose structures seem to be strictly involved in the adaptation to high temperatures. In this work, the complete structures of the major GL components from the cell envelope of the thermophilic bacterium Thermus thermophilus Samu-SA1 are presented. Protocols conventionally adopted for Gram-negative bacteria were used, and, for the first time, GL from Thermus were analyzed in their native form. Two GL and one phosphoglycolipid (PGL) were detected and characterized. The two GL, analyzed by nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, possessed the same tetrasaccharide structure linked to a glycerol unit or, alternatively, to a long-chain diol. Moreover, a PGL from Thermus was characterized for the first time, in which N-glyceroyl-heptadecaneamine was present. These molecules are chemically related to other GL from thermophile bacteria, in which they play a crucial role in the adaptation of cell membranes to heat.