In this study, the partial section of mitochondrial cytochrome b in 23 individuals of four Bactrocera cucurbitae geographic populations (Banna, Ruili, Yuanjiang, and Hekou)in Yunnan Province was sequenced, and the phylogenetic trees of all haplotypes were constructed by means of N-J method, using B. dorsalis, B. correct and B. tau as the out-group species. Of the 426 bp mitochondrial cytochrome b DNA sequences obtained, A% + T% was about 65.0%, four nucleotide sites were substituted, and no base composition of the sequences was inserted and lost. Five haplotypes were identified in all the sequences, with four showing polymorphism, and one shared. The genetic characters of the four populations were analyzed by Fst value and gene flow through Mega software. The Fst value was 0.16667-0.20000 (P > 0.05), and the Nm value was 2.00 - 2.50. The N-J tree suggested that four haplotypes constructed three clades. All results showed that the genetic differences presented among the four populations, but with low degrees. The main reason causing the genetic differences among the four populations was supposed to be related to genetic isolation and ecological adaptation.