BACKROUND: The melon fruit fly Bactrocera cucurbitae and the peach fruit fly Bactrocera zonata are serious pests, native to Asia, that have recently invaded Egypt. In Tunisia, no report of these species has yet been made, but pest risk data suggest that both Bactrocera species are likely to establish in other countries of the Mediterranean region. RESULTS: A quick method, based on a PCR-RFLP of the mitochondrial COI gene, has been developed to enhance species identification for quarantine purposes. The restriction enzymes Ddel and Xmnl were used to digest COI PCR products, enabling a rapid diagnosis of B. cucurbitae and B. zonata and their distinction from Tunisia's most devastating fruit pest, Ceratitis capitata. CONCLUSION: The simplicity and relatively low cost of this molecular approach will replace the need to rear immature stages through to adults for identification and will facilitate rapid quarantine decisions providing greater plant protection.