The root of Gentiana lutea L. is famous for its bitter properties and often used in alcoholic bitter beverages, food products, and traditional medicine to stimulate the appetite and improve digestion. This study presents the first report of an accurate, simple, fast, and reliable headspace solid phase microextraction (HS–SPME) method coupled to gas chromatography– mass spectrometry (GC–MS) for the analysis of the volatile profile of G. lutea roots. The developed method proved to be reliable to perform comparative analysis of the volatiles constituents from different populations of cultivated, wild, and commercial roots of G. lutea. HS–SPME/GC–MS analysis of 22 samples led to identification of 154 compounds belonging to terpenoids, alcohols, aldehydes, ketones, esters, alkanes, fatty acid derivatives, benzene derivatives, and others. The results proved that the composition of the volatile fraction of cultivated G. lutea is mostly similar to that of the wild samples but quite different with respect to commercial roots, and this was confirmed by principal component analysis (PCA).