Two full-length genes encoding different acetylcholinesterases (AChEs), designated as Ch-ace1 and Ch-ace2, were cloned from strains of the rice stem borer (Chilo suppressalis) susceptible and resistant to the organophosphate insecticide triazophos. Sequence analysis found an amino acid mutation A314S in Ch-ace1 (corresponding to A201 in Torpedo californica AChE) that was consistently associated with the occurrence of resistance. This mutation removed an MspA1 I restriction site from the wild type allele. An assay based on restriction fragment length polymorphism (RFLP) analysis was developed to diagnose A314S genotypes in field populations. Results showed a strong correlation between frequencies of the mutation and phenotypic levels of resistance to triazophos. The assay offers a prospect for rapid monitoring of resistance and assisting with the appropriate choice of insecticide for combating damage caused by C. suppressalis.