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Pentacyclic triterpenes have a diverse biological activity and they are promising targets for the medicinal applications in numerous disorders. The high amount of triterpenes is found in Lamiaceae and Ericaceae family; however, the new sources of these compounds are continuously being looked for. Different species of Carlina genus have been commonly used in traditional medicine in many countries and their pharmacological properties may be related to pentacyclic triterpenes; thus, the objective of this work was the analysis of various species of Carlina genus regarding the presence of triterpenic compounds and their distribution in different parts of plant. So far, the chemical study of these plants refers only to the determination of polyphenols and the essential oils composition. HPTLC was used as a screening method for qualitative investigations. The quantification of triterpenes was done using RP-HPLC-PDA on spherical and monolithic stationary phases. The identity of compounds was established by HR-APCI-MS and 1H NMR techniques. Our investigation reveals the occurrence of α-amyrin, β-amyrin, β-amyrin acetate, lupeol, lupeol acetate, betulinic, oleanolic and ursolic acid mostly in aboveground parts of the studied species. Carlina genus turned out to be a rich source of biologically active triterpenes.