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Chitosan is used worldwide as a food supplement and it is normally obtained from animal exoskeleton, mainly crustaceans. Chitosan produced from animals reportedly causes allergic reactions, thus only the equivalent deriving from fungi is permitted in winemaking. The OIV has issued specific methods to identify the biological origin of chitosan but these proved to be laborious and time consuming. To evaluate whether stable isotope ratios analysis could be a quicker and more effective tool to identify chitosan from fungi and from other origins, the H, C, N and O isotopic ratios of 35 samples deriving from various biosynthetic pathways are here presented. The Kruskal-Wallis test showed that δ13C and δ18O of the bulk samples were the most significant parameters (p < 0.0001), while a partial least squares discriminant analysis (PLS-DA) provided a prediction for abilities in cross-validation with a mean value of 100% both for calibration and validation subset. Diverse biosynthetic pathways during the formation of the precursor chitin influenced the isotopic composition of chitosan and allowed for an effective characterization of the product from fungi. Image 1 • In winemaking only fungi chitosan is allowed. • Biosynthetic origin of chitosan has an effect on isotopic ratios. • The isotopic ranges of chitosan from fungi and animal are different. [ABSTRACT FROM AUTHOR]