Cordyceps sinensis, a well-known and valued traditional Chinese medicine and usually also called as DongChongXiaCao (winter worm summer grass) in Chinese, is a composite consisting of the stromata of the fungus and the dead caterpillar. Cordyceps is commonly used to replenish the kidney and soothe the lung for the treatment of multiple diseases. Due to the rarity and upstanding curative effects of natural Cordyceps, several mycelial strains have been isolated from natural Cordyceps and manufactured in large quantities by fermentation technology, and they are commonly sold as health food products and/or drug. Cordyceps contains high amount of polysaccharides which are responsible for a lot of biological activities of Cordyceps. Therefore, analysis of polysaccharides from natural C. sinensis is helpful to control the quality of Cordyceps and cultured products. Firstly, to ensure the origin and quality of polysaccharides, a novel method, saccharide mapping, based on endo-carbohydrase hydrolysis followed by chromatographic analysis was established and successfully applied for discrimination of polysaccharides from 9 famous Traditional Chinese Medicines (TCMs) including Cordyceps. Subsequently, the method was further performed for characterization and comparison of polysaccharides from natural and cultured Cordyceps species depend on their chemical characteristics such as glycosidic linkages. Furthermore, water soluble polysaccharides from natural C. sinensis, fewly reported before, were isolated and purified, which was guided by antioxidant activity assay. Three polysaccharides named NCSP-I, NCSP-II and NCSP-III were obtained, and NCSP-III showed significant antioxidation activity. Besides, carbohydrates including free and polymeric carbohydrates in natural and cultured Cordyceps samples were also firstly determined by using stepwise pressurized liquid extraction (PLE) extraction and GC-MS analysis. In summary, a new saccahride mapping was established and proved to be useful for qualitative analysis of polysaccharides in TCM such as Cordyceps. The investigation on characteristics of polysaccharides and carbohydrates from Cordyceps is helpful for improving the quality control of Cordyceps.