Chitosan crystallization, performed by simple drying of an oligomer or polymer solution on a glass or mica surface, was studied at room temperature. Polarizing microscopy revealed the formation of two-dimensional spherulites that were shown to have a negative birefringence. After metal shadowing, electron microscopy investigations allowed the molecular arrangements of these structures to be described more precisely. X-ray and selected area electron diffraction experiments led to the identification of the crystalline structure which corresponds to the monoclinic chitosan hydrochloride. The presence of water in the crystalline lattice was also revealed.