Amur grayling, Thymallus grubii, is an important economic cold freshwater fish originally found in the Amur basin. Currently, suffering from loss of habitat and shrinking population size, T. grubii is restricted to the mountain river branches of the Amur basin. In order to assess the genetic diversity, population genetic structure and infer the evolutionary history within the species, we analysised the whole mitochondrial DNA control region (CR) of 95 individuals from 10 rivers in China, as well as 12 individuals from Ingoda/Onon and Bureya River throughout its distribution area. A total of 64 variable sites were observed and 45 haplotypes were identified excluding sites with gaps/missing data. Phylogenetic analysis was able to confidently predict two subclade topologies well supported by maximum-parsimony and Bayesian methods. However, basal branching patterns cannot be unambiguously estimated. Haplotypes from the mitochondrial clades displayed local homogeneity, implying a strong population structure within T. grubii. Analysis of molecular variance detected significant differences among the different geographical rivers, suggesting that T. grubii in each river should be managed and conserved separately.