Bactrocera dorsalis (Diptera: Tephritidae) is an important pest for many tropical and subtropical fruits. The fly is probably introduced in Yunnan, a southwestern province of China that shares borders with Vietnam, Laos and Myanmar. Depending on local environmental conditions, this species occurs either only in the most favorable seasons or year-round. To infer the genetic diversity and structure of the fly in the region, and to understand the relationships between the flies of year-round and seasonal areas, we analyzed 304 individuals from 14 populations using the mitochondrial cytochrome oxidase I gene (COI). The sampled populations were structured into four groups, probably isolated by the main natural barriers in Yunnan such as mountain ranges and rivers. Our data suggest either that B. dorsalis in Yunnan originated from multiple introductions events, even if the source populations still need to be identified; or that Yunnan is a natural origin of this species (i.e., that it is not invasive there). Finally, we found some evidences that the seasonal populations were colonized from nearby year-round populations.