Abstract: The distribution of Triplophysa yarkandensis is restricted in the Tarim River basin. Due to the gradual depletion of water resources, together with alien invasion and agricultural cultivation in Tarim River, the population size of T. yarkandensis has been diminishing and critically endangered. Changing this trajectory will require coordinated researches and conservation strategies to provide a better understanding of population genetics at large spatial scales. To accomplish it, eight populations of T. yarkandensis were collected from Qarqan River and Weigan River, and the genetic diversity, population structure and population dynamics were analyzed by sequencing the mitochondrial cytochrome b (Cytb) gene and control region (D-loop). Based on the Cytb and D-loop sequences, 41 and 51 haplotypes were detected, and the haplotype diversity/nucleotide diversity were 0.9247±0.0124/0.9776±0.0032 and 0.0065±0.0034/0.0115±0.0058, respectively, indicating a high genetic diversity. The AMOVA results showed that, the genetic variations mainly derived from intrapopulation (Cytb F_st=96.57%; D-loop F_st=95.25%), with only 3.43% (Cytb) and 4.75% (D-loop) from inter-populations. In addition, the low genetic distance and genetic differentiation index F_st between populations and no obvious geographical clustering and pedigree structure formatting according to the phylogenetic tree and network evolution diagram of haplotypes analyzing revealed a small degree of genetic differentiation among populations. Although there were potential genetic barriers and genetic differentiation between populations from Weigan River and Qarqan River, the genetic distance value was only around 0.05 (Cytb, F_ct=0.0540; D-loop, F_ct=0.0471). The results of neutrality test and nucleotide mismatch distribution plotting indicated that T. yarkandensis populations in Tarim River basin were relatively stable and no obvious population expansion occurred recently. To sum up, the genetic diversity of T. yarkandensis populations in Tarim River relative higher, but the genetic divergence was mainly from intrapopulation, and obvious gene flow between populations in the studied area was supported by our results. Thus, we suggest protecting it as one protection unit. These results provide valuable genetic data for developing scientific management strategies and directing future monitoring and utilization of the wild resource of T. yarkandensis in Tarim River basin.