Macrobrachium nipponense is a major farmed species freshwater shrimp in China and its genetic diversity is of high value in aquaculture. The construction of the Three Gorges Project has slowed down the water velocity in the Three Gorges Reservoir (TGR) and made the water more clear, which is more favourable for the survival and reproduction of M. nipponense. However, there are lack of studies on the genetic diversity of M. nipponense populations in the TGR. In this study, mitochondrial COI gene sequences were used as molecular marker to analyze the genetic diversity, genetic differentiation and historical demography of eight M. nipponense populations in the TGR. The results showed that the M. nipponense populations in TGR have high genetic diversity. There was some genetic differentiation between the four populations in lower reaches of the TGR and four populations in the upper reaches of the TGR. We also found that there was a significant correlation between genetic distance and geographical distance among the M. nipponense populations in the TGR. The M. nipponense population in the TGR has experienced population reduction in the recent year, which might be related to the ice age movement in the Little Ice Age and human activities. This study provides a theoretical basis for the rational development and protection of M. nipponense resources in the TGR.