Chamerion angustifolium (L.) Holub (syn. Epilobium angustifolium L., Onagraceae family) plants are utilized as a component of drugs, nutraceuticals and cosmetic products. The European Medicines Agency (EMA) aprooved E. angustifolium in traditional herbal medicinal products for treatment and alleviating symptoms related to Benign Prostatic Hyperplasia (BPH). Ch. angustifolium extracts have shown: anti-androgenic, anti-tumor, anti-inflammatory, analgesic, antioxidant and antimicrobial activities. Plants are a rich source of ellagitannins, flavonoids and phenolic acids, the herb also contains steroids, teriterpens and fatty acids. So far, phenols of this species have been widely studied while sterols have been neglected in this respect. Campesterol, cholesterol, stigmasterol and β-sitosterol and its derivatives have been identified in plants. Phytosterols are synthesized and accumulated in plant in vitro cultures, in this way in vitro cultures could be a an alternative source for the production of phytosterols. The aim of this study was to detrmine the content of campasterol, β-sitosterol and stigmasterol in Ch. angustifolium plants cultivated in vitro. The plants (shoots) grown on ½ MS medium supplemented with IAA (0.5 mg L^-1) and vitamin C (0.1 g L^-1) were collected after five weeks of culture and subjected to the HPLC-DAD anlysis. The mean content of campesterol, stigmasterol and β-sitosterol was: 216.06±82.74 mg/100 g, 464.93±69.56 mg/100 g, 156.08±49.13 mg 100 g^-1, respectively. The investigated genotypes differed in sterols content, particuraly in β-sitosterol: 69.79 – 222.49 mg 100 g^-1 DW. In this study, the effect of genotype on sterols accumulation under in vitro conditions was shown.