AbstractBackgroundThe two‐spotted spider mite (TSSM), Tetranychus urticae (Acari: Tetranychidae), is a cosmopolitan phytophagous pest in agriculture and horticulture. It has developed resistance to many acaricides by target‐site mutations. Understanding the status and evolution of resistant mutations in the field is essential for resistance management. Here, we applied a high‐throughput Kompetitive allele‐specific polymerase chain reaction (KASP) method for detecting six mutations conferring resistance to four acaricides of the TSSM. We genotyped 3274 female adults of TSSM from 43 populations collected across China in 2017, 2020, and 2021.ResultsThe KASP genotyping of 24 testing individuals showed 99% agreement with Sanger sequencing results. KASP assays showed that most populations had a high frequency of mutations conferring avermectin (G314D and G326E) and pyridaben (H92R) resistance. The frequency of mutation conferring bifenazate (A269V and G126S) and etoxazole (I1017F) resistance was relatively low. Multiple mutations were common in the TSSM, with 70.2% and 24.6% of individuals having 2–6 and 7–10 of 10 possible resistant alleles, respectively. No loci were linked in most populations among the six mutations, indicating the development of multiple resistance is mainly by independent selection. However, G314D and I1017F on the nuclear genome deviated from Hardy–Weinberg equilibrium in most populations, indicating significant selective pressure on TSSM populations by acaricides or fitness cost of the mutations in the absence of acaricide selection.ConclusionOur study revealed that the high frequency of TSSMs evolved multiple resistant mutations in population and individual levels by independent selection across China, alarming for managing multiple‐acaricides resistance. © 2023 Society of Chemical Industry.