Bronze gudgeon, Coreius heterodon, is an endemic and economically important fish in the Yangtze River, whose abundance has declined dramatically because of dam construction, overfishing, and water pollution. The Gezhouba and Three Gorges dams block connection of the bronze gudgeon populations above and below the dams. We collected bronze gudgeon from four sites in the mainstem of the Yangtze River, with one site above the dams and three sites below the dams, and studied genetic structure within and among the samples using 12 microsatellite DNA markers. Differences in indexes of genetic diversity were not significant among all the samples. No recent dramatic decrease of effective population size was inferred for all the samples using the population bottleneck test. Overall and pairwise genetic differentiation showed no significant genetic differentiation. Membership proportions of three genetic clusters inferred using assignment analysis were not significantly different among the samples. These results suggested that the genetic diversity and structure of bronze gudgeon were uniform across the samples. However, the Hardy-Weinberg equilibrium test, fixation index and linkage disequilibrium test indicated genetic subdivision of bronze gudgeon in the upper reach of the Three Gorges Dam. The present study and future studies including tributary samples will provide an important baseline of genetic diversity and population structure of bronze gudgeon in the Yangtze River, which is critical for monitoring and evaluating impacts of the large-scale dams on this species.