In the 21st century, the risk of the exotic pest being invaded is increasing due to the rise of trading activities. Oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae), has been considered to very destructive invader as it is highly intrusive and has a wide host range. In this study, 2,299 samples were collected from 88 locations in 12 countries by quarantine and investigation. Among them, 608 individuals were used for COI DNA barcoding analysis based on Neighbor Joining method with P-distance model. Population genetics analysis was conducted for 510 individuals selected from 47 locations of 12 Southeast Asian countries using 15 microsatellite markers. COI DNA barcoding results showed that B. dorsalis did not support any clade as a geographical isolation but almost indicated mixed populations of each country. Population genetics analysis showed similar pattern of genetic structure between neighboring countries across borders. Most domestic quarantine groups were more similar in genetic structure to Taiwan, China and Thailand in the order of appearance.