AbstractBACKGROUNDInsecticide resistance has emerged in various western flower thrips (WFT) populations across the world, threatening the efficiency of chemical control applications. Elucidation of insecticide resistance mechanisms at the molecular level provides markers for the development of diagnostics to monitor the trait and support evidence‐based resistance management.RESULTSTaqMan and Droplet Digital polymerase chain reaction (ddPCR) diagnostics were developed and validated, against Sanger sequencing, in individual and pooled WFT samples respectively, for the G275E mutation (nicotinic acetylcholine receptor α6 gene, nAChR α6) associated with resistance to nAChR allosteric modulators, site I (spinosyns); L1014F, T929I, T929C and T292V mutations (voltage‐gated sodium channel gene, vgsc) linked with pyrethroid resistance; and I1017M (chitin synthase 1 gene, chs1) conferring resistance to growth inhibitors affecting CHS1 (benzoylureas). The detection limits of ddPCR assays for mutant allelic frequencies (MAF) were in the range of 0.1%–0.2%. The assays were applied in nine WFT field populations from Crete, Greece. The G275E (MAF = 29.66%–100.0%), T929I and T929V (combined MAF = 100%), L1014F (MAF = 11.01%–37.29%), and I1017M (MAF = 17.74%–51.07%) mutations were present in all populations.CONCLUSIONThe molecular diagnostics panel that was developed in this study can facilitate the quick and sensitive resistance monitoring of WFT populations at the molecular level, to support evidence‐based insecticide resistance management strategies. © 2022 Society of Chemical Industry.