Rationale:Pesticide isomers are widely available in agricultural production and may vary widely in biological activity, potency, and toxicity. Chromatographic and mass spectrometric analysis of pesticide isomers is challenging due to structural similarities. Methods:Based on liquid chromatography-time of flight mass spectrometry (LC-Q-TOF/MS), identification of cis-trans isomeric pesticides was achieved through retention time, characteristic fragment ions, and relative abundance ratio. The cleavage pathways of six cis-trans isomers were elucidated through collision-induced dissociation (CID) to explain the origins of different fragment ions. The energy-resolved mass spectrometry combined with computational chemical density functional theory in terms of kinetics, thermodynamics, and bond lengths, the specific process of fragments were simulated to explain the reasons for the differences in characteristic fragment ions and abundance ratios. Results:Based on the above study, a high-resolution mass spectrometry method was developed for the separation and analysis of cis-trans isomers of pesticides in traditional Chinese medicine Radix Codonopsis, and six pesticide isomers were distinguished by retention time, product ions, and relative abundance ratios. The limits of quantification of the six pesticides were up to 10 μg/kg, and the linear ranges of them were 10-200 μg/kg, with the coefficients of determination (R ²)＞0.99, which demonstrated the good linearity of the six pesticides. The recoveries of the pesticides at the spiked concentrations of 10 μg/kg, 20 μg/kg, and 100 μg/kg reached 70-120% with the RSDs ≤20%. Conclusions:It was demonstrated that the application of the method was well-suited for accurate qualitative and quantitative analysis for each isomers with different structures which could avoid false negative results caused by ignoring another isomers effectively.