Polysaccharides from seven species of natural and cultured Cordyceps were firstly investigated and compared using saccharide mapping, partially acidic/enzymatic (α-amylase, β-glucanase and pectinase) digestion followed with polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and high performance thin layer chromatography (HPTLC) analysis, respectively, to obtain the comprehensive profiles of hydrolysates of the polysaccharides and their characters. The results showed that 1,4-α-d-glucosidic, 1,4-β-d-glucosidic and 1,4-α-d-galactosidic linkages were existed in natural and cultured Cordyceps sinensis, cultured Cordyceps militaris, natural Cordyceps gracilis and Cordyceps ciecadae. The similarity of polysaccharides from cultured C. militaris to natural C. sinensis was relatively high, which might contribute to the rational use of C. militaris. Moreover, different species of natural and cultured Cordyceps can be differentiated based on the saccharide mapping, which is helpful to well understand the structural characters of polysaccharides from different species of Cordyceps and to improve the quality control of polysaccharides in natural and cultured Cordyceps.