29 mitochondrial Dloop gene segments of Squaliobarbus curriculus from Yangtze River and Pearl River were amplified with PCR technique and sequenced. Analysis on Dloop of 599 bp showed that there were 24 unique sequences, 25 singleton, 39 parsiminfo, 81 mutation sites in all, including 50 transition sites, 14 transversion sites and 17 insert/lacuna sites. The content of A+T (68.8%) was much more than that of C+G (31.2%). The genetic diversities among the 5 water areas ranged from 0 to 6.03%. Haplotypic diversity(H) was 0.977 8，mean number of pairwise differences (K±SD) was 17.271 0, nucleotide diversity (π) was 0.029 7. F_ST values between WH and SS, XF, WZ populations, between YC and SS, XF, WZ populations showed significant genetic differences, and the one among all the other populations showed no strong genetic differences. Significant genetic differentiation among all five populations was found by analysis of molecular variance (AMOVA). According to the molecular phylogenetic tree constructed by NJ methods based on Dloop, the individuals of Yangtze River and Pearl River assembled in two branches respectively with high confidence. The obvious genetic differentiation was mainly attributed to the reproductive isolation caused by the geographical isolation between Yangtze River and Pearl River. But, for Yangtze River, the molecular phylogenetic tree was immingled because the intrapopulation and interpopulation genetic distances differed little. So it showed that there was no genetic differentiation in those from different water areas, which belonged to ‘Yangtze population’. It was also the case with ‘Pearl population’.