Presence and identity of secondary metabolites are two of the main components of lichen taxonomy. Aromatic compounds formed via the acetyl-polymalonate pathway are the most studied lichen substances. In addition, compounds derived from the mevalonic acid pathway (e.g., terpenes and steroids) are sometimes detected in the medulla. However, their identity and value as diagnostic characters in the genus Usnea are yet poorly understood despite the fact that they were mentioned in several taxonomical papers. We conclude that i) aside from the previously recognized polyphenolic compounds, carbohydrates and steroids are also detected in the medulla of some Usnea species; ii) the use of sulfuric anisaldehyde reagent greatly improves the detection of terpenes, carbohydrates and steroids compared with the sulfuric acid reagent routinely used in thin layer chromatography; iii) among carbohydrates, we detected arabitol and sucrose in the medulla; iv) steroids and terpenes remain unidentified and deserve further investigations.