[Objective] The present paper aims to explore the feasibility of DNA barcoding applied to the identification of flies in Yunnan province, China, and to set up DNA database for flies in Yunnan. [Method ] Diptera insects trapped by microbial attractants in Hekou county, Dali city, Jianchuan county, Mengzi city and Yuanjiang county of Yunnan province were identified by CO! gene sequence, and their genetic distances of families, genera and species taxa were compared. [Result] Forty-three species of insects belonging to 21 genera, 7 families including Drosophilidae, Tephritidae, Calliphoridae, Fanniidae, Muscidae, Sarcophgidae and Anthomyiidae, were collected in the 5 places in Yunnan. lntraspecific divergence ranged between 0. 002 and 0. 098, interspecific divergence average 0. 156. Phylogenetic tree showed that most of the same species gathered closely although some species were not in line with their morphology based on their sequences data. The average genetic distances among species of Drosophilidae, Tephritidae, Calliphoridae, Fanniidae, Muscidae, Sarcophgidae and Anthomyiidae were 0. 151, 0. 225, 0. 123, 0. 143, 0. 158, 0. 158 and 0. 103, respectively. The intraspecific genetic distances were 0. 006, 0. 025, 0. 012, 0. 013, 0. 011, 0. 006 and 0. 01, respectively. The genetic distances among 7 families were 25. 2, 10. 2, 10. 2, 11. 0, 14. 4, 27 . 3 and 10. 3 times of intraspecific genetic distances, respectively. [Conclusion] DNA barcoding based on CO/ gene can be used as a complementary means for morphological identification of some common flies in Yunnan province. [ABSTRACT FROM AUTHOR]