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Abstract Background Eye pigmentation genes have been utilized as visible markers for constructing genetic control prototypes in several insect vectors of human disease. Here, orthologs of two ommochrome pathway genes, kynurenine 3-hydroxylase (kmo) and cardinal, were investigated in Plutella xylostella, a globally distributed, economically important pest of Brassica crops. Results Both somatic mosaic and germline mutations were efficiently created using the CRISPR/Cas9 system, and null mutant strains of Pxkmo and Pxcardinal were obtained. A frame-shift mutation in Pxkmo caused yellow compound eyes at adult stage while an in-frame mutation lacking two amino acids resulted in a hypomorphic red eye phenotypes. In contrast, Pxcardinal-deficient moths with a frame-shift mutation exhibited yellow eye pigmentation in newly emerged adults which turned to red as the adults aged. Additionally, differences were observed in the coloration of larval ocelli, brains and testes in Pxkmo and Pxcardinal yellow-eye mutant lines. Conclusions Our work identifies the important roles of Pxkmo and Pxcardinal in P. xylostella eye pigmentation and provides tools for future genetic manipulation of this important crop pest.