Abstract In this work, we compared the patterns of polyphenol components from five varieties of cherries belonging to the family of Prunus avium (sweet cherry, 3 cultivars ) and Prunus cerasus (tart cherry, 2 cultivars ). The total phenol content was determined by the Folin–Ciocalteu method and the in vitro antioxidant activity by the ORAC and ABTS assays. Colorless polyphenols and anthocyanins were characterized by reversed phase-HPLC-diode array detection (DAD) and mass spectrometry (MS) combined techniques, including MALDI-TOF MS and nano-HPLC-ESI-Q-TOF-MS/MS. The polyphenol profiles were dominated by chlorogenic acid isomers and quercetin glycosides, though they substantially differed from each other. Interestingly, kaempferol-3- O -rutinoside exclusively marked the P. cerasus varieties. In all the samples, the most abundant anthocyanins were cyanidin (Cy) derivatives, mainly Cy-3- O -glucosylrutinoside, whereas Cy-3- O -xylosylrutinoside was exclusively found in tart cherries. The characteristic expression of branched tri-glycosides supports the taxonomic lineage of P. cerasus as an interspecific hybrid of P. avium and probably P. fruticosa . The anthocyanin profiles appeared both species- and cultivar -specific. Thus, after opportune validation over a larger number of varieties and biological replicates, the anthocyanin composition has potential to be exploited for classification purposes and for tracing back the cherry biotype in processed products ( e.g. cherry juice, jam, flavored yogurt).