Abstract: Mylopharyngodon piceus is one of the four major fish and plays a very important role in ensuring the supply of aquatic products in China. In recent years, due to the destruction of wild resources and the intervention of artificial factors, the wild resources of M. piceus have been destroyed, and there is also a lack of corresponding data on the selection and breeding of improved M. piceus. In order to master the distribution characteristics and reveal the genetic variation of M. piceus germplasm resources, this study designed primers for 271 samples of mitochondrial COⅠ region of 9 M. piceus populations and conducted PCR amplification. The genetic diversity and the evolutionary relationships among populations were analyzed by softwares. The results showed that GC content was lower than AT content in the COⅠ gene region of 9 populations with the length of 1003 bp. A total of 6 mutation sites and 7 haplotypes were detected, and Hap4 haplotypes were distributed in 9 populations. AMOVA showed that 90.33% of the variation of 271 M. piceus samples came from within and 9.67% from between populations. The total nucleotide diversity (π) of M. piceus was low, ranging from 0.00109 to 0.00244. Haplotype diversity (H_d) was high, ranging from 0.403 to 0.847. The coefficient of genetic differentiation (F_st) ranged from –0.0033 to 0.23445, and remained low to high, mainly concentrated in low and moderate differentiation. The results showed that the Foshan population may have experienced bottleneck effect, and the other 8 populations may have experienced rapid population expansion. The results of this study reveal the genetic background of Chinese M. piceus germplasm resources, which can provide important reference for the conservation and innovative utilization.