Sixteen cytochrome P450 gene fragments from the red imported fire ant were isolated from a fire ant EST library. These P450 fragments exhibited significant protein homologies with cytochrome P450 families 4, 6, 9, and 319, with the similarities arranged from 35 to 72%. The P450 protein signature motif, FXXGXRXCXG, is present in all the P450 fragments. Northern blot analysis was performed to compare expression levels of these P450 genes for different development stages (1st +2nd instar larva, 3rd+4th instar larva, pupa, and adult) and castes of fire ants. We found that the expression of most of these P450 genes were developmentally and caste specifically regulated in red imported fire ants. mRNA levels of these P450s were overexpressed in the workers with no significant differences between big workers and small workers. Their caste-specific overexpression suggests the importance of these P450 genes in workers of the red imported fire ant. The functions of the P450s identified in the fire ant as metabolism of endogenous compounds in workers, response to queens’ chemical signals, as well as involved in the caste differentiation merit the consideration.