The composition of fatty acids (FAs) is known to discriminate families within genera of bacteria, fungi, octocorals and algae. Here, we applied the more sensitive gas chromatographic (GC) analysis to investigate the taxonomic value of using FA composition to discriminate 15 species of lichen, including three chlorolichens (two Cladonia species and Stereocaulon scutelligerum), nine tripartite lichens (Stereocaulon species) and three cyanolichens (Lichina pygmaea, Collema cristatum, Peltigera membranacea). One macroscopic cyanobacterium (Nostoc sp.), corresponding to the photobiont partner of Peltigera and Collema cyanolichens, was included for comparison. Five lipid extraction methods were evaluated, using Stereocaulon scutelligerum. Shaking at 80 °C with chloroform/methanol (2:1, v/v) was the most efficient and reproducible method. The total FA composition of the 16 species was compared, using GC coupled with flame ionization detection or mass spectrometry. A statistical hierarchical cluster analysis was used to determine the similarity between the FA profiles, based on total, saturated, unsaturated and branched-chain FAs (BCFAs). The BCFA profile allowed a distinction between the Stereocaulon species and the cyanolichens, in contrast to all the other types of FAs. A detailed characterization of eight FA subclasses, provided by the GC analysis, suggested a preferential pathway for the biogenesis of unsaturated FAs through desaturases, which was especially favoured in the cyanolichens.