BackgroundInvasive Australian populations of redlegged earth mite, Halotydeus destructor (Tucker), are evolving increasing organophosphate resistance. In addition to the canonical ace gene, the target gene of organophosphates, the H. destructor genome contains many radiated ace‐like genes that vary in copy number and amino acid sequence. In this work, we characterise copy number and target‐site mutation variation at the canonical ace and ace‐like genes and test for potential associations with organophosphate insensitivity. This was achieved through comparisons of whole‐genome pool‐seq data from alive and dead mites following organophosphate exposure.ResultsA combination of increased copy number and target‐site mutations at the canonical ace was associated with organophosphate insensitivity in H. destructor. Resistant populations were segregating for G119S, A201S, F331Y at the canonical ace. A subset of populations also had copy numbers of canonical ace > 2, which potentially helps overexpress proteins carrying these target‐site mutations. Haplotypes possessing different copy numbers and target‐site mutations of the canonical ace gene may be under selection across H. destructor populations. We also detected some evidence that increases in copy number of radiated ace‐like genes are associated with organophosphate insensitivity, which might suggest potential roles in sequestration or breakdown of organophosphates.ConclusionDifferent combinations of target‐site mutations and (or) copy number variation in the canonical ace and ace‐like genes may provide non‐convergent ways for H. destructor to respond to organophosphate selection. However, these changes may only play a partial role in organophosphate insensitivity, which appears to have a polygenic architecture. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.