Two novel P450 cDNAs, CYP6A36 and CYP6A37, were isolated from house-flies. Putative protein sequences deduced from the cDNA sequences shared 58% identity. Predicted protein sequences of CYP6A36 and CYP6A37 from pyrethroid-resistant ALHF house-flies were identical to their corresponding orthologues in susceptible aabys flies. Expression of CYP6A36 was developmentally regulated with significant overexpression in ALHF compared with susceptible CS flies. Overexpression of CYP6A36 was detected in ALHF abdomen, where the primary detoxification organs of the midgut and fat body are located. CYP6A37, however, was expressed in all tested life stages, with no significant difference in expression between ALHF and CS. Genetic linkage analysis located CYP6A36 on autosome 5; overexpression of CYP6A36 was linked to the factors on autosomes 1 and 2, corresponding to the linkage of P450-mediated resistance in ALHF. This evidence suggests the importance of CYP6A36 in detoxification of insecticides and evolution of insecticide resistance in ALHF.