The development of hydroelectricity in the Nujiang River would have adverse impacts on the populations of Glyptothorax zanaensis. In order to assess the genetic diversity and differentiation of this species, we sequenced the cytochrome b gene of the mitochondria in 102 individuals of the fish collected from 6 sampling sites (Gongshan, Gudeng and Lushui in the Nujiang Prefectural District and Daojie, Mengnuo and Mucheng in the Baoshan Municipal District). A total of 87 variation sites were detected in the fragment of 1 137 bp in length, with which the 102 samples were defined as 36 haplotypes. The haplotype diversity (h) and the nucleotide diversity (pi) of total samples were 0.851+/-0.028 and 0.01356+/-0.0008, respectively. Therefore, the genetic diversity of G. zanaensis was relatively low. However, the genetic diversity of the Nujiang population was significantly higher than that of the Baoshan population. The pairwise Fst value between the populations (0.475-0.846) was higher than that within the population (0.002-0.108), which implied that the Fst value was positively related to geographic distance. Analysis of molecular variance (AMOVA) showed that the genetic differentiation between the populations and within the populations were 53.65% and 46.35%, respectively. The fixation index (Fst value) was 0.5365, indicating that there existed significant differentiation between the Nujiang population and the Baoshan population. The phylogentic tree and networks of the haplotypes of G. zanaensis showed that there were two separate lineages: the Nujiang lineage and the Baoshan lineage. Each lineage represents at least one separated management unit, or belongs to an evolutionary significant unit. It was suggested that in the construction of hydroelectric projects the measures for protecting G. zanaensis should be adopted in fully considering the populations of G. zanaensis and the status quo of their population structure to avoid the occurrence of gene exchange among populations.