Mythimna separata (Walker) (Lepidoptera: Noctuidae) is one of the most destructive phytophagous pests. Here we present detailed instructions to achieve successful application of CRISPR/Cas9‐mediated genome editing on M. separata. We optimized procedures, including egg collection and incubation. We further successfully conducted microinjection on the washed eggs by applying water on the egg surface. We then evaluated the effects of injection timing on both egg viability and genome editing efficiency using NPC1b as the target gene. We observed that Cas9 protein injection had significantly less effect on the viability of the eggs at the earlier stage (<2 h old) than at later stages, although no significant effect was observed on the postembryonic development between different egg ages. All larvae were edited when the eggs were <6 h old, and the editing efficiency decreased when older eggs were used. Moreover, mutants were detected among G1 progenies, which demonstrated that the mutation generated by the Cas9/sgRNAs system on M. separata was heritable. As far as we know, this is the first report of CRISPR/Cas9 for this important pest. The successful application of genome editing on M. separata is essential to understanding its biology and developing novel control methods. Mythimna separata (Lepidoptera: Noctuidae) is an important agricultural pest. We report detailed instructions to achieve the application of CRISPR/Cas9–mediated genome editing on M. separata, including egg collection and incubation, embryo microinjection, and injection timing. This is the first report of CRISPR/Cas9‐mediated genome editing in M. separata, and the information is essential to conduct molecular research on the biology of this important pest.