Three polysaccharide fractions (PS-I, PS-II, and PS-III) were isolated from the aqueous extract of a hybrid mushroom obtained through backcross mating of a somatic hybrid mushroom PfloVv12 (Sterile line) with Volvariella volvacea. PfloVv12 was obtained through protoplast fusion of Pleurotus florida and V. volvacea. PS-I was identified as 1,6-β glucan. PS-II and PS-III were identified as mannoglucogalactan but differing in molecular weights only. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, and NMR experiment ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) the structures of these polysaccharides were established as;