The Nu River Project comprised of 13-dam cascade is one of the biggest hydropower development projects yet conceived in China. However, hydropower development will inevitably result in the local ecological environment and biological material impact, particularly the effects on fish. Glyptothorax zainaensis is weak in swimming with suction cup and is extremely sensitive in the hydrological characteristics and water quality changes, so that it can be considered as a water quality monitor of indicator organism. Currently, with the need for conservation, fishery restoration and so on, there is a great demand for genetic information on Glyptothorax zainaensis. Fourty microsatellite loci were isolated, characterized and evaluated from Glyptothorax zainaensis. A total of 91 different alleles were observed in the 303 wild Glyptothorax zainaensis, and the percentage of polymorphic bands ranged from 41.8% to 66.7%, with an average of 90.1%. The genetic diversity value was significantly huge with the expected heterozygosity ranging from 0.578 to 0.706, the observed heterozygosity value ranging from 0.923 to 0.980 and PIC value ranging from 0.418 to 0.644, which showed that the wild Glyptothorax zainaensis population had a high level of genetic diversity. Analysis of molecular variance (AMOVA) and pairwise Fst showed a significant level of genetic differentiation (P < 0.01) among the populations accounting for 40.2% of the total variance. Additionally, results derived from different statistical techniques with different software packages included cluster analysis on NJ tree. Apart from such molecular approaches, we opposed four ways to strengthen the effective resources protection.