Abstract: To investigate the enantioselective behavior of chiral fungicide penthiopyrad in tomato under field and greenhouse conditions, a residue analytical method was developed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the determination of enantiomers of penthiopyrad in tomatoes based on an optimized QuEChERS approach. The proposed method was used for studying the behavior of residues, dissipation and accumulation of penthiopyrad enantiomers in tomato system under two cultivate patterns. The results indicated that at the range of 0.002-1 mg/L, the linearity of the penthiopyrad enantiomers was good with R² > 0.99. At the spiked levels of 0.005, 0.1 and 1 mg/kg, the average recoveries of the R-(−)-PEN and S-(+)-PEN in different matrices were 77%-98% and 81%-101%, and the relative standard deviations (RSDs) were 1.6%-5.1%and 1.6%-4.2%, respectively. The limit of quantification (LOQ) was 0.005 mg/kg. The degradation rates of the residues were < 30% in tomato samples stored at −20 ℃ for 100 d. The dissipation trend in tomato was fitted with the first-order kinetic equation in both the open field and greenhouse planting modes, and the correlation coefficients (r) were 0.9315-0.9400. The half-lives of R-(−)-PEN and S-(+)-PEN were 5.43 and 5.10 d in the open field, and were 8.09 and7.80 d in the greenhouse modes, respectively. The dissipation of S-(+)-PEN was faster than that of R-(−)-PEN, and the enantiomers of penthiopyrad was accumulated in tomato after repeated spraying. The results of residue distribution also showed that penthiopyrad enantiomers were mainly distributed in the leaves of tomato, and the concentration in tomato leaves, stems and fruits in greenhouse was higher than that in open field. On the 5th days after the last application, the residues of the penthiopyrad were lower than the maximum residue limit (MRL) in China, and the residues of its metabolite (PAM) were lower than the LOQ. The results indicated that the application of penthiopyrad on tomatoes in accordance with the recommended method complies with Good Agricultural Practices in China and meets the basic requirements for food safety.