Abstract O?Mannosylation is a vital protein modification involved in brain and muscle development whereas the biological relevance of O-mannosyl glycans has remained largely unknown owing to the lack of structurally defined glycoforms. An efficient scaffold synthesis/enzymatic extension (SSEE) strategy was developed to prepare such structures by combining gram-scale convergent chemical syntheses of three scaffolds and strictly controlled sequential enzymatic extension catalyzed by glycosyltransferases. In total, 45 O-mannosyl glycans were obtained, covering the majority of identified mammalian structures. Subsequent glycan microarray analysis revealed fine specificities of glycan-binding proteins and specific antisera.