Hevein, a protein found in Hevea brasiliensis, has a CRD domain, which is known to bind chitin and GlcNAc-containing oligosaccharides. By using NMR and molecular modeling as major tools we have demonstrated that trisaccharides containing GalNAc and ManNAc residues are also recognized by hevein domains. Thus far unknown trisaccharides GlcNAcβ(1→4)GlcNAcβ(1→4)ManNAc (1) and GalNAcβ(1→4)GlcNAcβ(1→4)ManNAc (2) were synthesized with the use of β-N-acetylhexosaminidase from Aspergillus oryzae. This method is based on the rather unique phenomenon that some fungal β-N-acetylhexosaminidases cannot hydrolyze disaccharide GlcNAcβ(1→4)ManNAc (5) contrary to chitobiose GlcNAcβ(1→4)GlcNAc (4) that is cleaved and, therefore, cannot be used as an acceptor for further transglycosylation. Both trisaccharides 1 and 2 were prepared by transglycosylation from disaccharidic acceptor 5 in good yields ranging from 35% to 40%. Our observations strongly indicate that the present nature of the modifications of chitotriose (GlcNAcβ(1→4)GlcNAcβ(1→4)GlcNAc, 3) at either the non-reducing end (GalNAc instead of GlcNAc) or at the reducing end (ManNAc instead of GlcNAc) do not modify the mode of binding of the trisaccharide to hevein. The association constant values indicate that chitotriose (3) binding is better than that of 1 and 2, and that the binding of 1 (with ManNAc at the reducing end) is favored with respect to that of 2 (with ManNAc at the reducing end with a non-reducing GalNAc moiety).