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Lepidopteran pests are of major economic importance to macadamia growers worldwide. In South Africa, four species have been associated with macadamia nut damage. Detailed information regarding species composition of the borer larvae in the nuts and the genetic diversity of these species is not however available.Lepidoptera obtained from nuts from the KwaZulu‐Natal, Mpumalanga and Limpopo provinces were identified based on COI sequencing.Thaumatotibia batrachopa represented 95% of the larvae collected in damaged nuts across all growing regions.The population genetic diversity and structure of the dominant species were determined using a 650 bp section of the mtDNA cytochrome oxidase I gene to construct a parsimony network.Rapid diagnostic tools were developed, which included a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay for the identification of all four species and species‐specific primers for T. batrachopa and T. leucotreta.Population studies of T. batrachopa showed a dominant COI haplotype present in all growing regions, although several unique haplotypes were also present in each region. Overall, the high haplotype diversity observed in this study is in agreement with that of a native population. The populations between the growing regions are, however, not clearly separated and might reflect recent gene flow. [ABSTRACT FROM AUTHOR]

Simple Summary: Stink bugs inflict extensive damage leading to significant yield and economic loss in the South African macadamia industry. There is currently a need for alternative control strategies to replace the reliance on chemical control in South Africa. Accurate identification and knowledge of species composition are important to inform these management practices. In this study, we identified stink bug species associated with macadamia orchards in the three main growing regions of South Africa. This was performed based on morphology and DNA barcoding. A total of 21 stink bug species were found in macadamia orchards, and Bathycoelia distincta was the dominant species found. A group of Boerias spp. were found to be dominant in KwaZulu-Natal, and this is the first report of these species associated with macadamia. Evidence of cryptic species diversity was also found within Pseudatelus raptorius and an unidentified Boerias sp. (Boerias sp. 1). Species composition fluctuated over three growing seasons and between growing regions, highlighting the need for ongoing monitoring of these important pest species. The DNA barcode database developed in this study will be valuable for future monitoring, identifications and the implementation of informed management strategies. Stink bugs are major pests of macadamia in South Africa. Accurate identification and knowledge of species composition are important to inform management practices. The overall aims of this study were to identify stink bug species from macadamia orchards in South Africa using morphology, and to establish a DNA database based on the cytochrome c oxidase subunit 1 gene region. A total of 21 stink bug species were found in macadamia orchards in KwaZulu-Natal, Limpopo and Mpumalanga provinces. Bathycoelia distincta Distant, 1878, was the dominant species throughout all three growing regions. Two unidentified species of Boerias Kirkaldy, 1909, here designated as Boerias sp. 1 and Boerias sp. 2, were the second and third most abundant species found in KwaZulu-Natal. No species of Boerias has previously been reported in association with macadamia. Evidence of a cryptic third species of Boerias was also found. Species composition fluctuated over three growing seasons in Limpopo and differed between the three growing regions during the 2019–2020 season, highlighting the need for ongoing monitoring of these important pest species. The DNA barcode database developed in this study will be valuable for future monitoring and identifications, including cryptic or polymorphic stink bug species and different life stages. [ABSTRACT FROM AUTHOR]