Cicerbita alpina (L.) Wallr, is a perennial alpine plant and a member of the Asteraceae family, typically found at altitudes above 1000 meters in the Italian Alps. Although previously utilized primarily as a local delicacy, recent studies have revealed strong antiparasitic activity through in vitro experiments. In Europe, numerous chemical drugs employed to combat nematodes — helminths that infest the digestive tract of livestock — are banned due to their environmental harm or show only reduced efficiency because of the development of resistance. Consequently, there is a growing demand for new alternative anthelmintic treatments in agricultural practices. Specialized metabolites found in the extracts of C. alpina could offer a sustainable and biological alternative to chemical drugs, specifically for nematode control. For this purpose, a unique germplasm collection originating from eight distinct natural populations in the Italian Alps was analyzed for its chemical diversity using state-of-the-art targeted LC-MS/MS spectrometry, including quantification based on multiple reaction monitoring. The predominant metabolites identified from the species were the caffeic acid derivatives chicoric acid, chlorogenic acid, and 3. 5-dicaffeoylquinic acid, the sesquiterpene lactone derivative 8-O-acetyl-15-ß-D-glucopyranosyl lactucin and the flavone glycosides, apigenin-7-O-glucoside and luteolin-7-O-glucoside, alongside their precursors apigenin and luteolin, respectively. Additionally, the genetic diversity of eighty individual plants within the germplasm collection was evaluated using ten DNA molecular markers (Simple Sequence Repeats), successfully transferred from two closely related species (Cichorium intybus and Tanacetum parthenium). This investigation unveiled a significant range of genetic diversity within the examined populations, resulting in the establishment of three distinct genetic groups. The findings were further correlated with the original ecological environment and local climate conditions spanning a biennial period, indicating substantial variations among the different accessions and the intricate interplay between genetic background and environmental factors. These results could serve as a basis for future domestication of the species through plant breeding programs ensuring product quality, but also facilitating the cultivation of C. alpina in more diverse geographic regions.