We examined genetic variation in the Oriental fruit fly, Bactrocera dorsalis (Hendel), using six populations in two regions of Yunnan Province, China, to determine the distribution and likely mechanism for the dispersal of this fly. A 501-bp portion of the mitochondrial cytochrome oxidase gene was sequenced from a minimum of eight individuals from each population, and 43 haplotypes were observed in the six Bactrocera dorsalis populations. When comparing the genetic diversity of populations in the northern and southern regions, which differ with respect to elevation, climate and plant phenology, we found a significantly greater haplotype diversity in the southern region (permutation test; P < 0.05), suggesting that the northern populations, those at Kunming and Qujing, probably originated from somewhere in the southern region. FST and number of pairwise differences revealed a high level of differentiation between the Panxi population and the other populations (permutation test; P < 0.05). Although the difference was marginally insignificant, the Shuitang population seemed to have differentiated from both northern populations. The Mantel test did not detect any isolation due to geographic distance. An amova analysis found that 2.56% of the variance was caused by the Panxi population. Haplotype network analysis showed that none of the six populations had a specific genetic lineage. Together, these analyses suggest that long-distance dispersal has occurred for this species, and the species most probably took advantage of both a mountain pass and prevailing air currents. The Panxi population was significantly isolated from the others, probably because of its distinguishing habitat features, host plants or the recent reduction of the population size.