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The habitat of Chinese mitten crabs (CMCs, Eriocheir sinensis) has significant impact on their flavor and market price, and the characteristic flavor of CMCs produced in Yangcheng Lake in the lower reach of the Yangtze River in China is the most sought after by consumers. In this study, the differences in the intestinal microbiome and flavor amino acids, nucleotides, and organic acids in CMCs cultivated in Yangcheng Lake and Gucheng Lake (also in the lower reach of the Yangtze River) using the same model were compared. The results showed that CMCs cultivated in different lakes contained specific microbiota, with significantly different composition and abundance of the dominant microbiota. Although remarkable differences were noted in the contents of flavor amino acids, nucleotides, and lactic acid among different tissues and genders of CMCs, only some of these indicators varied between CMCs of different habitats, with respect to the distinctive flavor of CMCs. Thus, the difference in the intestinal microbiota of CMCs from different habitats may be related to the variation in the distinctive flavor of CMCs.