The vinegar or pomace fly, Zaprionus tuberculatus Malloch, is native to the Afrotropical region but has recently colonized new areas in Turkey, Italy, Malta, Romania, and Greece. This invasive species was first detected in 2012 in Adana on banana and peach baits and is considered a potential agricultural pest in fig production in Turkey. However, the European and Mediterranean Plant Organization reports that no direct association of the fly with fruit damage has been documented so far because specimens were generally caught in traps. Specimens of Zaprionus sp. were monitored during surveys for cases of Drosophila suzukii (Matsumura) and Ceratitis capitata (Wiedemann) damaging experimental pomegranate and fig fields in Canakkale, where we performed the field study. To better understand the species identification, a partial sequence of the mitochondrial cytochrome oxidase I (COI) gene region was used in this study. The barcode primer pairs were used to amplify the partial COI gene. PCR products were visualized and then directly sequenced. No insertion or deletion was observed in the 637-bp COI gene region. The average nucleotide frequencies of T, A, C, and G were observed as 39.1%, 29.6%, 15.2%, and 16.1%, respectively. Intraspecific pairwise divergence was low at 0.15%. Nucleotide sequences indicated that there were 2 substitutions, an A -> G at 343 and a C -> T at 592 positions, respectively. In conclusion, this study presents the first molecular evidence and presence of Z. tuberculatus in Canakkale, Turkey, which can help in resolving morphologic ambiguities.