The composition of fatty acids (FAs) in gametophyte samples of 20 Siberian bryophyte species from four orders of mosses and four orders of liverworts collected in relatively cold months (April and/or October) was examined. FA profiles were obtained using gas chromatography. Thirty-seven FAs were found, from 12:0 to 26:0; they included mono-, polyunsaturated (PUFAs) and rare FAs, such as 22:5n–3 and two acetylenic FAs, 6a,9,12–18:3 and 6a,9,12,15–18:4 (dicranin). Acetylenic FAs were found in all examined species of the Bryales and Dicranales orders, dicranin being the predominant FA. The role of particular PUFAs in mosses and liverworts is discussed. Multivariate discriminant analysis (MDA) was performed to determine whether FAs can be used in the chemotaxonomy of bryophytes. Based on the MDA results, FA composition is related to the taxonomic status of species. Thus, several individual FAs were identified as chemotaxonomic markers at the level of bryophyte orders. These were 18:3n–3; 18:4n–3; 6a,9,12–18:3; 6a,9,12,15–18:4; 20:4n–3 and EPA in mosses and 16:3n–3; 16:2n–6; 18:2n–6; 18:3n–3 and EPA in liverworts. These findings indicate that further research into bryophyte FA profiles can shed light on phylogenetic relationships within this group of plants and the evolution of their metabolic pathways.