Nine shikonin pigments: shikonin (S), acetylshikonin (AS), propionylshikonin (PS), isobutyrylshikonin (IBS), tiglylshikonin (TS), 3,3-dimethylacrylshikonin (DAS), angelylshikonin (ANS), 2-methyl-n-butyrylshikonin (MBS) and isovalerylshikonin (IVS) were identified in the root epidermis of Echium italicum L. for the first time. A new thin-layer chromatographic (TLC) method for the separation of enantiomers alkannin and shikonin proved only shikonin after saponification of the root extract, and was afterwards esterified with the corresponding acyl chloride to acquire seven standard compounds (all except ANS). The developed isocratic high-peformance liquid chromatographic (HPLC) methods with VIS and mass spectrometry (MS) detection, allowed for the first time simultaneous separation of all nine compounds with similar structures including positional and geometric isomers in a short time. Structures of the main five compounds (AS, IBS, ANS, MBS, IVS) isolated from the extract by a new semi-preparative HPLC on C18 have additionally been confirmed by (1)H and (13)C nuclear magnetic resonance spectra, which were reported for AS and MBS for the first time.