The house fly, Musca domestica L., is one of the important medical and veterinary pests and the most common insects in the human habitat which has developed resistance to most synthetic insecticides. This pest in the livestock buildings has caused annoyance and leads to a decrease in productivity. The house fly is a mechanical vector of more than 100 different pathogens such as bacteria, protozoa, viruses and nematodes. The pyrethroids insecticides are one of the most important insecticides used to control the house fly. In this study, the resistance to two pyrethroid insecticides, cypermethrin and deltamethrin was evaluated in the house fly populations collected from Koohrang (Chaharmahal and Bakhtiari), Namfar dairy (Falavarjan-Isfahan) and Fazil dairy (Mobarake-Isfahan). The collected pupae were transferred to the breeding room and after 2-3 generations on artificial diet, bioassays using a topical application were performed on same-aged female flies (3- to 7- days). The LD50 values of cypermethrin in the Koohrang, Namfar and Fazil populations were 2.56, 241.9 and 461.4 ng a.i./fly, respectively. The LD50 values for deltamethrin in the Koohrang, Namfar and Fazil populations were 1.1, 50.2 and 174 ng a.i./fly, respectively. The results of bioassay showed that the resistance ratio to cypermethrin for Namfar and Fazil populations were 94 and 180-fold comparing to Koohrang population, respectively, and for deltamethrin were 45 and 158-fold. The detoxify enzymes activity esterases, glutathione S-transferase and cytochrome P450-dependent monooxygenases, were measured in three populations. The results of enzyme activity assays indicated that there are no significant differences between esterases activity in three tested populations. However, significant differences were found in the glutathione S-transferase and cytochrome P450-dependent monooxygenases activity between Koohrang population with Namfar and Fazil populations. Therefore, the pressure of pyrethroid insecticides leads to resistance in the house fly to cypermethrin, deltamethrin. Considering the rapid resistance development in the house fly to insecticides, it can be suggested that a rotation of insecticides with the different mode of actions and different chemical groups should be used in an integrated management program against the house fly.