AbstractBACKGROUNDOfficial stable isotope databases, based on the analysis of (D/H)I ethanol, (D/H)II ethanol, δ13Cethanol and δ18Owater of wine, are an indispensable tool for establishing the limits beyond which the mislabeling or the addition of sugar and/or water in wine production can be detected. The present study investigates, for the first time, whether the use of hybrid varieties instead of European Vitis vinifera for wine production can have an impact on the stable isotope ratios.RESULTSThe analyses were performed by isotope ratio mass spectrometry and site‐specific natural isotope fractionation by nuclear magnetic resonance, in accordance with the official methods of the International Organization of Grapes and Wine. The comparison shows the tendency of some stable isotope ratios of hybrid varieties, in particular (D/H)I, to deviate from the regional averages of the V. vinifera samples. Notably, Baron, Monarch and Regent showed significantly different values at one of the two sampling sites. Particularly high δ13C values characterize Helios compared to other hybrid varieties.CONCLUSIONFor the first time, and from an isotopic point of view, the present study investigates the wine obtained from hybrid varieties, showing that further attention should be paid to their interpretation, on the basis of the database established according to the European Regulation 2018/273. © 2022 Society of Chemical Industry.