AbstractChitosanases can be used to produce partially acetylated chitosan oligosaccharides (paCOS) for different applications, provided they are thoroughly characterized. However, recent studies indicate that the established classification system for chitosanases is too simplistic. Here, we apply a highly sensitive method for quantitatively sequencing paCOS to reassess the substrate specificities of the best-characterized class I–III chitosanases. The enzymes’ abilities to cleave bonds at GlcNAc residues positioned at subsite (−1) or (+1), on which the classification system is based, vary especially when the substrates have different fractions of acetylation (F _A ). Conflicts with the recent classification are observed at higher F _A , which were not investigated in prior specificity determinations. Initial analyses of pectin-degrading enzymes reveal that classifications of other polysaccharide-degrading enzymes should also be critically reassessed. Based on our results, we tentatively suggest a chitosanase classification system which is based on specificities and preferences of subsites (−2) to (+2).