Introduction - The segregation between the genera Gentians and Gentianella among the Gentianaceae family is poorly defined. In order to clarify the classification of these genera, some researchers have tried to incorporate data about the chemical constitution, but this has not yet been achieved in a comprehensive way. Objective - To develop a fast and reproducible analytical method for the observation of characteristic fingerprints of secondary metabolites of each genus. Methodology - Seven species were investigated, three Gentianella and four Gentiana selected for their close taxonomic links within each genus. Ten xanthones previously isolated from one of these species were used as chemotaxonomic markers. A UPLC/ESI-TOF-MS method was developed to analyse the methanolic extracts. Results - The UPLC/TOF-MS provided clear metabolic fingerprints and elemental composition of the compounds. The profiles of the three Gentianella species were strikingly similar. On the contrary, metabolic profiles of Gentians species were very different from the Gentianella chromatograms and also from each other. Several compounds were unique to each genus and therefore could be used as biomarkers. Conclusion - UPLC/TOF-MS can be applied as a chemotaxonomic tool for the rapid screening of Gentianaceae species and for the distinction between closely related taxa from the Gentianaceae family.