Glutathione S-transferases (GSTs) play central roles in phase II detoxification of both xenobiotics (drugs, insecticides, and herbicides) and endogenous compounds in almost all living organisms. In this study, we successfully purified the GSTs from the citrus red mite, Panonychus citri, by affinity chromatography on Glutathione Sepharose 4B and compared the biochemical characterizations of the purified GSTs from three field populations [beibei (BB), wanzhou (WZ), and zhongxian (ZX)]. SDS-PAGE revealed that the molecular weight of GSTs from three populations consisted of two subunits of 27.3 and 26.1 kDa. The specific activity of the purified GSTs from the WZ and ZX populations was increased 1.5- and 3.8-fold, respectively, compared with the BB population. Accordingly, the pyridaben susceptibility of WZ and ZX populations was less compared with BB population. Kinetic analyses showed that the WZ and ZX populations had higher substrate specificity compared with the BB population based on the values of k (cat) and k (cat) /K (m) to both reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). The in vitro inhibition studies of GSTs indicated that the I (50) values of pyridaben from WZ and ZX populations of P. citri expressed 1.6- and 4.4-fold decreases, respectively, compared to the I (50) value of pyridaben from the BB population. In conclusion, all evidence suggested that the purified GSTs may partially contribute to the susceptibility of acaricide pyridaben in field populations of P. citri.