Cytochrome P450s constitute the largest gene superfamily found in nature and are critical for detoxification and/or activation of xenobiotics and for metabolizing endogenous compounds. To better understand the physiological functions of P450s in the house fly, a medically and economically important insect species, a comprehensive effort has been made to deciphering P450 genes and their functions in this insect species. Here, we report more than 20 P450 gene fragments cloned and sequenced from house flies. These cDNA fragments exhibit significant protein homologies with cytochrome P450 families 4, 6, and 9 with similarities ranged from 48% to 84%. The P450 protein signature motif, FXXGXRXCXG, is present in all the P450 fragments. Northern blot analysis was performed to investigate expression patterns of these P450 genes in different development stages, male and females, resistant and susceptible flies, and the response to permethrin treatment. The possible roles of these P450s identified in the house fly have been discussed.