Cytochrome P450s (CYP450s) are known to detoxify insecticides in diamondback moth (DBM), Plutella xylostella. Knockdown bioassay with piperonyl butoxide indicated that the enhanced activity of CYP450 is associated with pyrethroid resistance mechanism in a cypermethrin-resistant strain (JJ) of DBM. To investigate the CYP450-mediated resistance mechanism on a molecular level, first we isolated Cyp450 genes from DBM by homology-probing PCRs using the degenerate primers designed from the subfamilies of Cyp4 and Cyp6B, which are generally known to be involved in insecticide resistance in a variety of insects. A total of 7 different Cyp genes were isolated. Six genes were classified to belong to Cyp4 family, and one to Cyp6B by sequence comparison. The six Cyp4-like genes were designated as Cyp4-I~VI on the basis of their relative frequencies (48.4, 17.7, 16.1, 11.3, 3.2, and 3.2%, respectively). The Cyp4-II, -III, and -IV genes were determined to be 6~20-fold more transcribed constitutively in the JJ strain as judged by quantitative real-time PCR analysis, implying their role in resistance. The last one gene belonging to Cyp6B was slightly more transcribed in the JJ strain. Pretreatment of sub-lethal dose of cypermethrin to the JJ strain slightly increased the transcription of Cyp4-II and -VI, suggesting that they might be inducible. In summary, the three Cyp4 genes constitutively over-expressed in the JJ strain are likely play a main role whereas two other Cyp4 genes tentatively inducible by cypermethrin a supporting role in the pyrethroid reisistance of DBM.