Abstract: In order to scientifically assess the germplasm resource status of Gobio huanghensis and further strengthen its germplasm resource conservation, this study was conducted on the basis of three consecutive years of resource investigation in the Ningxia section of the Yellow River. At the same time, samples from five different geographic populations in Qingtongxia, including Nanchangtan Village, Yuding Township, Meijiawan Village, Yueyahu Township and Hongyazi Township, were collected at a fixed point, and the genetic diversity and phylogenetic relationship were investigated based on mitochondrial cox1 gene sequence and bioinformatics analysis. The results showed that 162 polymorphic loci were detected in the 1 428 bp cox1 gene sequence region, and 26 haplotypes were defined. Among the five groups, the Nanchangtan Village group had "low H_d and low P_i"（H_d: 0.468; P_i: 0.000 62）, other groups showed high H_d and high P_i（H_d: 0.721～0.840; P_i: 0.009 18～0.035 88）, and there was significant genetic differentiation between the upper and lower populations of Qingtongxia（P＜0.05）, especially the Nanchangtan Village and Yuding Township populations and the Yueyahu populations reached the population differentiation level. Phylogenetic analysis showed that Gobio huanghensis was the closest relative to Saurogobio dabryi. In summary, based on the results of resource surveys over the years, it is speculated that Gobio huang-hensis forms obvious population geographic barrier due to Qingtongxia Dam. Therefore, it is suggested to divide Gobio huanghensis into two geographic populations above and below Qingtongxia Dam. On the basis of strengthening in situ protection, artificial intervention measures such as artificial breeding, breeding and release can be adopted to promote gene exchange of germplasm resources above and below the dam to effectively restore and protect the precious germplasm resources of Gobio Huanghensis.