The diamondback moth, Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae), is one of the most important pests of cruciferous crops in Iran and is controlled mostly by fenvalerate. The susceptibility to fenvalerate was investigated for four populations of P. xylostella. Bioassay results indicated significant differences among the populations tested. The highest level of resistance to fenvalerate was obtained for the Khuzestan (Khz) population (resistance ratio = 9.5). Survival was suppressed by diethyl maleate, piperonylbutoxide, and triphenyl phosphate, which confirmed that resistance to fenvalerate is caused by glutathione S-transferases (GSTs), mixed function oxidases, and esterases, respectively. Up to 8.6-, 2-, 2.7-, and 1.75-fold increases in GSTs, esterase (α-naphthylacerate and β-naphthyl acetate as substrate), and cytochrome P450 monooxygenases activities in resistant strains when compared with the susceptible one, were observed, respectively. The expression of six GST genes of P. xylostella including GSTs₁, GSTe₂, GSTe₄, GSTo₄, GSTd₄, and GSTd₅ were analyzed. The quantitative PCR analysis showed that three of the PxGSTs had the highest expression levels in the Khz population. Two of the GSTs (GSTd4 and PxGSTe2) exhibited highest expression level in both Khz and Alborz (Alb) populations. Therefore, PxGST genes were involved in fenvalerate resistance in P. xylostella. Overall, the mechanisms of insecticide resistance in diamondback moth populations in four regions of Iran were related to GST, esterase, and cytochrome P450 monooxygenase activities.