Five guaianolides, including two pairs of isomers, from a Hungarian taxon of the Achillea millefolium group were characterized analytically. Different chromatographic systems on TLC and HPLC were developed for the analysis of these compounds. TLC of leucodin, 8alpha-angeloxy-leucodin, achillin, 8alpha-angeloxy-achillin and desacetylmatricarin was performed on silica gel using dichloromethaneacetone and cyclohexane-ethylacetate mixtures as mobile phases. HPLC on stationary phases as LiChrospher RP2, LiChrospher RP8, LiChrospher RP18e, Hypersil BDS C18 and Aquasil C18 required isocratic and gradient systems with different methanol-water mixtures as mobile phases. The presented RF values and retention times allow the identification of the respective 2-oxo-guaianolides which are marker substances for certain non-proazulene containing species. Their TLC and HPLC fingerprints are compared to those of proazulene containing species and are relevant for quality control.