Abstract: This study aimed at investigating the genetic differentiation and gene flow among geographic populations of Curculio bimaculatus. The partial sequences of mtDNA COI gene in 9 geographic populations of C.bimaculatus were sequenced and analyzed in Yunnan. The molecular variance, the haplotype differentiation, the genetic diversity, gene flow were analyzed. The results showed a total of 79 COI gene with 32 variable sites and 34 haplotypes were obtained and were 693 bp, including 1 haplotype shared by 9 population. The haplotype diversity index (H_d) was 0.901 01, the total fixztion index (F_st) was 0.132 82, the total genetic differentiation coefficient (G_st) was 0.052 55 and the gene flow (N_m) was 9.01. The Tajima′s D test for all populations or each single population of C.bimaculatus did not reach any significant level, which suggest that there might not have been a recent population expansion and population stability. The results of AMOVA analysis demonstrated that genetic differentiation of C.bimaculatus mainly occurred within populations. The Mantel test disclosed that the genetic distance among populations was correlated with the geographic distance. The haplotype network and neighbor-joinjng phylogenetic tree showed that the haplotypes are distributed in different clades and no obvious geographical structure had been formed. This indicated that the high level of gene exchange did exist in different geographical populations.