Chitosans, a family of ß-(1,4)-linked, partially N-acetylated polyglucosamines, are considered to be among the most versatile and most promising functional biopolymers. Chemical analysis and bioactivity studies revealed that the functionalities of chitosans strongly depend on the polymers' degree of polymerization and fraction of acetylation. More recently, the pattern of acetylation ( PA) has been proposed as another important parameter to influence functionalities of chitosans. We therefore carried out studies on the acetylation pattern of chitosan polymers produced by three recombinant fungal chitin deacetylases (CDAs) originating from different species, namely, Podospora anserina, Puccinia graminis f. sp. tritici, and Pestalotiopsis sp. We analyzed the chitosans by 1H NMR, 13C NMR, and SEC-MALS and established new methods for PA analysis based on enzymatic mass spectrometric fingerprinting and in silico simulations. Our studies strongly indicate that the different CDAs indeed produce chitosans with different PA. Finally, Zimm plot analysis revealed that enzymatically treated polymers differ with respect to their second virial coefficient and radius of gyration indicating an influence of PA on polymer-solvent interactions.