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Nettle (Urtica dioica L.) root is a well-known source of valuable phytosterols, however scientific data regarding their composition are scarce. Therefore, this study aimed to examine the profile of phytosterols and triterpenes in nettle root (12 samples) harvested in three different climate regions and to provide more detailed information on the profile of nettle root lipophilic extract. Nettle root samples were extracted using n-hexane, while detection and quantification of phytosterols and triterpenes was achieved by GC–MS. Average content of total sterols was 0.80–0.86 g kg−1 dry weight and total pentacyclic triterpenes (PT) of 0.0048–0.0070 g kg−1. A total of 10 sterols and 3 PT were detected. The dominant sterol in all samples was β-sitosterol, followed by campesterol and stigmasterol. Lower quantities of Δ7 sterol - citrostadienol were detected. The dominant PT in root samples from warmer regions was β-amyrin acetate, while samples from the colder region showed the predominance of β-amyrin.