Abstract The diamondback moth, Plutella xylostella , is a global pest of cruciferous vegetables. Abamectin resistance in a field population of P. xylostella was introgressed into the susceptible Roth strain. The resulting introgression strain Roth‐Abm showed 11 000‐fold resistance to abamectin compared with Roth. An A309V substitution at the N‐terminus of the third transmembrane helix (M3) of the glutamate‐gated chloride channel of P. xylostella ( PxGluCl ) was identified in Roth‐Abm. The frequency of the V309 allele of PxGluCl was 94.7% in Roth‐Abm, whereas no such allele was detected in Roth. A subpopulation of Roth‐Abm was kept without abamectin selection for 20 generations to produce a revertant strain, Roth‐Abm‐D. Abamectin resistance in Roth‐Abm‐D declined to 1150‐fold compared with Roth, with the V309 allele frequency decreased to 9.6%. After treatment of the Roth‐Abm‐D strain with 80 mg/l abamectin the V309 allele frequency in the survivors increased to 55%. This demonstrates that the A309V mutation in PxGluCl is strongly associated with a 10‐fold increase in abamectin resistance in Roth‐Abm relative to Roth‐Abm‐D. Homology modelling and automated ligand docking results suggest that the A309V substitution allosterically modifies the abamectin‐binding site, as opposed to directly eliminating a key binding contact. Other resistance mechanisms to abamectin in Roth‐Abm are discussed besides the A309V mutation of PxGluCl .