The bird cherry-oat aphid, Rhopalosiphum padi (L.), is an insect pest that persistently attacks wheat crops worldwide. Glutathione S-transferases (GSTs) are important detoxification enzymes that play roles in insecticide resistance. In this study, we identified two GST genes (RpGSTS1 and RpGSTS2) from R. padi. Phylogenetic analysis indicated that the genes are associated with the sigma class of insect GSTs. The RpGSTS1 and RpGSTS2 contain nine α-helices and five β-sheets connected by loops, and had 60 and 50% homology with the 3D structure of the Blattella germanica GST5. We tested the toxicity of chlorpyrifos, imidacloprid, isoprocarb, sulfoxaflor, and λ-cyhalothrin to R. padi, and found that the toxicity of five insecticides to the aphid varied. The detoxification activity of GSTs and the expression patterns of RpGSTS1 and RpGSTS2 after insecticide treatments were also analyzed. Compared to the control, the GST activity was increased by 23, 18.5, 13, and 11.5% in aphids treated by LC50 concentrations of chlorpyrifos, isoprocarb, imidacloprid, and sulfoxaflor, respectively. Exposure to different chemical insecticides showed different effects on the expression of RpGSTS1 and RpGSTS2. These results indicate that RpGSTS1 and RpGSTS2 have unique biochemical characteristics and may play roles in resistance to insecticides in R. padi.