Chitosan is a natural polysaccharide which has been authorized for oenological practices for the treatment of musts and wines. This authorization is limited to chitosan of fungal origin while that of crustacean origin is prohibited. To guarantee its origin, a method based on the measurement of the stable isotope ratios (SIR) of carbonδ13C, nitrogenδ15N, oxygenδ18O and hydrogenδ2H of chitosan has been recently proposed without indicating the threshold authenticity limits of these parameters which, for the first time, were estimated in this paper. In addition, on part of the samples analysed through SIR, Fourier transform infrared spectrometry (FTIR) and thermogravimetric analysis (TGA) were performed as simple and rapid discrimination methods due to limited technological resources. Samples havingδ13C values above −14.2‰ and below −125.1‰ can be considered as authentic fungal chitosan without needing to analyse other parameters. If theδ13C value falls between −25.1‰ and −24.9‰, it is necessary to proceed further with the evaluation of the parameterδ15N, which must be above +2.7‰. Samples havingδ18O values lower than +25.3‰ can be considered as authentic fungal chitosan. The combination of maximum degradation temperatures (obtained using TGA) and peak areas of Amide I and NH2/Amide II (obtained using FTIR) also allows the discrimination between the two origins of the polysaccharide. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) based on TGA, FTIR and SIR data successfully distributed the tested samples into informative clusters. Therefore, we present the technologies described as part of a robust analytical strategy for the correct identification of chitosan samples from crustaceans or fungi.