The diamondback moth, Plutella xylostella is one of the most distributed and damaging pest of cruciferous crops in Iran, and is controlled almost by pyrethroids. One of this insecticide which is used for controlling this pest is fenvalerate. Some population of P. xylostella have become resistant to fenvalerate due to intensive use of this insecticide. Up to now, there is no information about the mechanisms of resistance in this pest population to fenvalerate which can help us to resistance management of this serious pest. The toxicity of fenvalerate in four populations from different parts of Iran (Tehran, Alborz, Khoozestan and Isfahan) was determined using leaf dipping bioassay method. The bioassay results showed significant difference in susceptibility to fenvalerate among the populations. Resistance ratio of different populations to the susceptible one ranged from 3.1 to 9.5. Synergist experiments was conducted using piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP) to determine whether mixed function oxidases, glutathione S-transferases and esterases were involved in resistance mechanisms, respectively. The level of resistance to insecticide was suppressed by PBO, DEM and TPP which confirmed that resistance to fenvalerate is mainly caused by detoxification enzymes. In order to determine the glutathione S-transferases, cytochrome P450 monooxygenase and esterase activities the methods of Van Asperen (1962), Habig et al. (1976) and De Sousa et al. (1995) were used with some modification, respectively. Cytochrome P450 monooxygenases activity increased 1.75 fold in resistant strain comparing with susceptible one. Up to 8.6- fold and 2-fold increase in glutathione S-transferase (GST) and esterase activities in resistant strains compared with the susceptible one was observed respectively. The real-time quantitative PCR (q RT PCR) reactions were conducted to show the expression level of GSTs. The changes in P. xyllostella GST gene expression levels exposed to fenvalerate indicate that they play important role in resistance to this insecticide. Overall, this study showed that the mechanisms of insecticide resistance in dimondback moth populations in four regions in Iran were related to cytochrome P450 monooxygenases, GST and esterase. This data help us to detect the mechanisms involved in resistance of P. xyllostella to fenvalerate and subsequently resistance management of this important pest.