Neosalanx taihuensis is an annual fish with commercial importance. This species is endemic to China, occurring in inland waters affiliated to the eastern-flowing rivers southern to the Yellow River, e. g. the rivers of Yellow, Huaihe, Yangtze, Ou and Pearl etc. Among these river basins, the Yangtze River tests to be the most important region for this species with the highest fishery stocks and the most populations, and thus to be the core area for its conservation and sustainable exploitation. In the near decades, however, N. taihuensis decreased severely with most of the natural populations extinguished economically or even ecologically. The local population extinction infers that the species' genetic diversity must have suffered bad influence and some genetic types must have lost as result. To test this presumption, we conducted an icefish-specific investigation in the mid-lower reaches of Yangtze River during August, 2006. Totally eight populations were sampled and used in the followed experiment on their genetic diversity. Based on the gene diversity of mitochondrial cytochrome oxidase subunit I(COI), we mainly focus on two points:1) to find out the genetic diversity in single population and among populations for understanding the genetic status of wild N. taihuensis in the river basin, 2) to check the suitability of COI gene for DNA barcoding study in the family of Salangidae. We selected 132 specimens, representing eight different geographic populations in the mid-lower Yangtze River basin, and analyzed their base sequences of COI gene. Among the 630 bp nucleotide sequence of COI gene, eight(1.27%) was variation sites, contributing to eight gene haplotypes. Although the number of gene haplotype and the nucleotide diversity index(π=0.00112 ± 0.00204) were not high, haplotype diversity(h) was fairly rich(h=0.576 ± 0.036) in the populations at river basin scale. Taking spatial distribution pattern into account, most gene haplotypes distributed in spatially narrow region with quite low occurrence incidences of 12.5% or 25.0%, occurred in one or two lakes respectively. Haplotype richness in each population varied significantly from each other, with a high fixation index. The least haplotypes occurred in the two highly isolated lakes of Dongting and Liangzi, and the highest haplotype richness occurred in the two lakes with icefish introduction histories. This result indicates that a high inter-population variation existed in the studied region. So we concluded that wild populations of N. taihuensis have suffered great ecological isolation, although they are geographically near to each other. Comparing with several other salangid fishes, e. g. Protosalanx hyalocranius, Hemisalanx brachyrostralis and N. oligodontis, the inter-specific genetic distance was 4.443%, amounting to some 35 times of that in N. taihuensis populations(0.125%). Compared with other fish taxa, such as Coilia, Culter, Oncorhynchus and Salmo, the intra-specific genetic distance was much smaller within N. taihuensis populations(< 1%). In conclusion, the COI gene is good and suitable to DNA barcode study in species identifying and systematic study, and can be useful in the assessment, monitoring and conservation management of icefish genetic diversity.