Context: Flavonoids are regarded as essential ingredients for the anti-inflammatory activity of Epilobium angustifolium L. (Onagraceae). The metabolic profiles of medicinal plants vary aside from genetic determination due to the influence by external factors. Objectives: This study evaluates the influence of altitudinal variation on the content of flavonoids in E. angustifolium in two consecutive growing periods. Materials and methods: Aerial and herbaceous plant materials were collected at three different altitudes (800, 1000, and 1500 m) during two collection periods. Plant samples (11-13 samples per altitude and year) were extracted with methanol using accelerated solvent extraction (ASE). Identification and quantification of the constituents were achieved by chromatographic means of HPLC-PDA and LC-PDA-MS analyses. Results: Rising concentrations of flavonol 3-O-glycosides could be detected with increasing altitude. The content of the major compound, quercetin 3-O-glucuronide, ranged from 4.4 ± 2.05 (800 m) to 4.9 ± 1.03 (1000 m) and up to 6.6 ± 1.14 mg/g (1500 m). The total amount of flavonol 3-O-glycosides in 73 analyzed samples ranged from 10.7 ± 1.37 up to 17.3 ± 1.99 mg/g. Discussion: Quercetin-3-O-glucuronide can be considered as a potential marker for the increased production of flavonols in herbal parts of E. angustifolium at higher altitudinal levels. Conclusion: The study confirms that environmental factors at higher altitude result in elevated levels of flavonols in aerial plant tissues of E. angustifolium. Specific factors for influencing the flavonoid content have to be clarified in further studies.