For further realizing the genetic diversity and provide experimental data of germplasm resources for Triplophysa brevicauda in the upper reaches of Lancang River in Yunnan Province, 77 individuals including 4 geographic populations (Biaocun, Yezhi, Lidi and Wu Nonglong) were analyzed. After sequencing, the total length of 921 bp of D-loop control region were obtained, and 22 variable sites (including 8 singleton variable sites and 14 parsimony informative sites) and 21 haplotypes were identified in whole populations, respectively; the haplotypic diversity (H_d) and nucleotide diversity (π) of Triplophysa brevicauda in the upper reaches of Lancang River was 0.837-0.942 and 0.005 16-0.005 72, respectively; among them, the highest H_d was identified in Lidi population, the lowest H_d was identified in Biaocun population, and a genetic character of "high H_d low π" was presented; The results of AMOVA indicated that most of genetic variation (97.91%) came from within population and little of genetic variation (2.09%) was came from between populations; no significant genetic differentiation was detected between the comparison groups (F_st=-0.021 3, P=0.805); the farthest genetic distance (0.005 67) of Triplophysa brevicauda was identified between Biaocun and Lidi population, and the closest genetic distance (0.005 11) was found between Yezhi and Wu Nonglong population; no recent population expansion event happened in the evolutionary history of Triplophysa brevicauda, which was proved by the evidence of non-poisson distribution of nucleotide mismatch and insignificant neutral test result (Tajima's D=0.624, P=0.737;Fu's F_s=-0.669, P=0.377). The results showed relatively abundant genetic diversity and low level of genetic differentiationo of Triplophysa brevicauda in the upper reaches of Lancang River.