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Polysaccharides from 39 batches of cultured Cordyceps militaris fruiting body produced in China were investigated and compared using saccharide mapping, partial acidic/enzymatic (α-amylase and β-glucanase) digestion followed by polysaccharide analysis using carbohydrate gel electrophoresis analysis (PACE). The results showed that 1,4-α-D-glucosidic and 1,4-β-D-glucosidic linkages existed in cultivated C. militaris and natural Cordyceps sinensis. There were relatively high similarities among the polysaccharides from different batches of C. militaris, and also between the polysaccharides from C. militaris and natural C. sinensis, which suggested that C. militaris could, at least partially, substitute natural C. sinensis. Furthermore, the results showed that the nitric oxide released from RAW 264.7 cells, induced by polysaccharides from C. militaris and C. sinensis, correlated with their 1,4-α-D-glucosidic and 1,4-β-D-glucosidic linkages, which is beneficial for better understanding of their structure–bioactivity relationship, and helpful to improve pharmacological activity-based quality control of polysaccharides in C. militaris.