Fruit flies (Diptera: Tephritidae) are significant insect pests of many commercially important fruits and vegetables. Therefore, rapid and accurate species identification methods are required for the regulation, management and quarantine of these pests. In this study, we examined the efficiency of mitochondrial cytochrome oxidase I sequences for species identification of fruit flies in Thailand. Data analyses based on 42 fruit fly taxa revealed moderate performance of this genetic marker. There were 14 taxa that have no barcode gap and thus could not be identified unambiguously to species by this methodology. Taxonomic uncertainty, inadequate variation of the marker and misidentifications of specimens deposited in the public database are the most likely factors explaining unsuccessful identification. DNA barcodes also revealed cryptic diversity in five taxa (, , , , ). These species require further taxonomic investigation of if they are different cryptic taxa or are indications of geographic structuring of within single species.