Aedes aegypti, an important vector in the transmission of human diseases, has developed resistance to two commonly used classes of insecticides, pyrethroids and organophosphates, in populations worldwide. This study examined sensitivity/resistance to chlorpyrifos, fenitrothion, malathion, deltamethrin, permethrin, and β-cyfluthrin, along with possible metabolic detoxification and target site insensitivity, in three Aedes aegypti mosquito strains. The resistant strain (PR) had developed high levels of resistance to all three pyrethroid insecticides compared to a susceptible population, with 6, 500-, 3200- and 17,000-fold resistance to permethrin, β-cyfluthrin, and deltamethrin, respectively. A newly emerged Ae. aegypti population collected from St. Augustine, Florida (AeStA) showed elevated levels of resistance to malathion (12-fold) and permethrin (25-fold). Synergists DEF (S,S,S,-tributyl phosphorotrithioate) and DEM (diethyl maleate) showed no or minor effects on insecticide resistance in both the AeStA and PR^G20strains, but PBO (piperonyl butoxide) completely abolished resistance to both malathion and permethrin in AeStA and partially suppressed resistance in PR. The voltage-gated sodium channel sequences were examined to explore the mechanism that only partially inhibited the suppression of resistance to PBO in PR. Two mutations, V1016G/I and F1534C substitutions, both of which are associated with the development of pyrethroid resistance, were identified in the PR^G20 strain but not in AeStA. These results suggest that while cytochrome P450 mediated detoxification may not be solely responsible, it is the major mechanism governing the development of resistance in AeStA. Both P450 mediated detoxification and target site insensitivity through the mutations in the voltage-gated sodium channel contribute to the high levels of resistance in the PR^G20 strain.