Significance Insect sex is determined genetically and shows high diversity among different species. The silkworm, Bombyx mori , has a WZ/ZZ sex chromosome system seen in lepidopteran insects in which the females are heterogametic. Sex separation methods are critical for commercial aspects of rearing silkworms, and genetic-sexing systems also could serve as the basis for adaptation to sterile insect techniques for pest lepidopteran species. Here, we describe a W chromosome-based, genetic-sexing system combining transcriptional activator-like effector nucleases and CRISPR/Cas9 technologies in B. mori . Development of silkworm strains with ubiquitous female-specific fluorescence for convenient genetic sorting or complete female-specific embryonic lethality for male-only rearing provides a successful example of targeting an insect sex chromosome with genome editing tools, which should assist future sterile insect technique development for pest insects.