Using mitochondrial DNA (mtDNA) Cyt b gene and D-loop control region as molecular markers, genetic diversities of 60 Pseudecheneis sulcata samples from two populations in the upper Lilong section and the lower Motuo section of the Yarlung Zangbo River Grand Canyon were studied. The effective sequence length of the combination gene was 1 893 bp, including 1 060 bp Cyt b gene and 833 bp D-loop control region. The results showed that the haplotype diversity values (Hd) of both Lilong and Motuo populations were high (0.701 and 0.761), and the nucleotide diversity values (π) were low (0.001 00 and 0.001 09). A high frequency of Hap1 and Hap2 was observed in both populations, which were presumed to be ancestral haplotypes. Meanwhile, there were 5 and 6 unique haplotypes in Lilong and Motuo populations, respectively, and they were not shared between the two populations. Analysis of molecular variance (AMOVA) showed that the genetic variation mainly originated from within the population, with moderate genetic differentiation between populations (Fst = 0.090 44, P < 0.05). Neutral test (Tajima''s D and Fu''s Fs) and nucleotide mismatch analysis (SSD and Hir) revealed that Pseudecheneis sulcata had experienced population expansion. This study indicated that there were barriers in gene flow between the two populations of P. sulcata. The ecological barriers such as altitude difference and hydrological situation of the Yarlung Zangbo Grand Canyon may be the main reasons for hindering the migration and communication in P. sulcata.