Fruit flies that belong to the genus (Diptera: Tephritidae) pose significant threats as invasive pests of agricultural crops in Asia and sub-Saharan Africa. The intensification of transboundary trade in fresh horticultural produce has increased the risk of introducing invasive species such as fruit flies, more so through the inadvertent transport of their immature developmental stages. Such immature stages of fruit flies belonging to the Tephritidae family are frequently intercepted at the international borders worldwide and are unable to be identified to the species level using morphological characteristics. Molecular identification using mitochondrial Cytochrome Oxidase I () gene has proven to be quite useful, as they are not constrained by developmental stages, sex, or colour morphs of the pest species in question. Also, real-time PCR-based species-specific assays offer quicker turnaround time since they do not require any post-PCR procedures. This study evaluated the utility of a real-time PCR assay based on the gene region to identify from other Tephritid species. The developed real-time PCR assay provides a swift and precise way of discriminating between these highly invasive pest species during an interception event for rapid decision making. High specificity, having no cross-reactions with closely related Tephritids, and sensitivity of the developed assay will be extremely useful in discriminating from other closely related fruit fly species. -specific real-time PCR developed in this study is appropriate for organizations that carry out routine diagnostics to facilitate fresh produce imports and exports. Our assay is fully optimized for rapid deployment at international borders, offering reliable detection of the target species regardless of developmental stage, sex, or geographic origins.