The Lower Gangetic Plains are India's central horticultural crop production regions. Fruit flies (Diptera: Tephritidae) are a significant insect pest that significantly hinders the effective crop production of various commercially valuable fruit and vegetable crops. Therefore, rapid and precise species identification techniques are essential to controlling, managing, and quarantining these pests. Furthermore, reliably identifying fruit flies is exceedingly challenging due to their striking resemblances in morphological characters. In this work, we investigated the effectiveness of mitochondrial cytochrome c oxidase I (COI) gene sequences for identifying seven fruit fly species, involving only the genus Bactrocera from the lower Gangetic Plains in eastern India. The sequences of the studied species showed 99%–100% similarity with sequences from within and outside the country. COI barcodes were able to distinguish between species, with K2P intraspecific genetic divergences ranging from 0-1% and K2P interspecific genetic divergences ranging from 3.65-28.46%, thus maintaining a proper barcode gap. Phylogenetic analyses were performed, and the results showed moderate to high supported monophyly among species. Therefore, COI barcodes have proven a highly successful alternative for quickly identifying fruit flies. This is also the first account of molecular identification of the previously reported tephritid fruit flies infesting fruits and vegetables in eastern India's agriculturally important lower Gangetic Plains.