Abstract
Introduction Apple production in Kazakhstan is highly vulnerable to damage caused by fruit-infesting tortricid pests, particularly codling moths (Lepidoptera: Tortricidae). Accurate and rapid species identification is essential for effective pest management and phytosanitary monitoring in apple orchards. Methods Field surveys were conducted in apple orchards of southern and southeastern Kazakhstan to investigate the codling moth complex. Collected specimens were identified morphologically and confirmed by sequencing a mitochondrial cytochrome c oxidase subunit I (COI) region. Comparative COI sequence analysis was used to develop a multiplex quantitative polymerase chain reaction assay employing Minor Groove Binder hydrolysis probes for simultaneous detection and differentiation of Cydia pomonella and Grapholita molesta . Results Field surveys identified C. pomonella and G. molesta , with C. pomonella accounting for 83.3% of collected specimens. COI sequencing enabled reliable discrimination between the two species. The multiplex assay demonstrated high specificity, produced no false-positive amplification in non-target species, and achieved 100% detection down to 5 pg DNA per reaction. Discussion The developed COI-based multiplex quantitative polymerase chain reaction assay provides a rapid, sensitive, and reliable tool for simultaneous identification of C. pomonella and G. molesta . It is suitable for phytosanitary monitoring, early detection, and management of quarantine-relevant tortricid pests in Kazakhstan.