The oriental fruit fly, Bactrocera dorsalis Hendel, is one of the most destructive pest insects of tropical and subtropical fruits and vegetables. It is thought to be an introduced species in Yunnan Province, China, where it causes severe damage. Depending on the latitude, the fly occurs year-round or only during the warm season. To assess the genetic diversity of the fly and to understand the relative isolation of its populations in this mountainous region, we conducted an analysis of population genetic structure using mitochondrial cytochrome oxidase (COI) gene sequences. Twenty-eight haplotypes were detected among 37 individuals with up to 13 mutations between haplotypes. Within-population diversity was high, and genetic distances between haplotypes reached 2.2%. The haplotype network showed that many haplotypes were missing in the sampled populations. Intraspecific variability in Bactrocera dorsalis was thus high in Yunnan. The data suggested either a longer residence of the fly in Yunnan than recognized previously or a recurrent colonization process from different origins. One population, namely Ruili, was significantly isolated from the others, probably because of geographic barriers to gene flow. This population seemed to be in a contact zone with flies originating from surrounding regions. In contrast, some populations separated by >300 km were not significantly structured. We suggest that the insects engage in long range dispersal, most probably taking advantage of prevailing air currents. The data also suggested that the region of Kunming, where the fly only occurs seasonally, is recolonized each year by migrating flies from several southern regions.