Abstract: In order to further enhance the efficiency of untargeted screening for hazardous substances in edible fungi, based on liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) combined with QuEChERS pretreatment, a high-throughput rapid screening platform for pesticides and their metabolites in edible fungi was established by acquiring the first-order accurate precursor mass values and secondary fragment spectral information of target analytes. The qualitative identification criteria were defined for the platform, including a precursor ion mass deviation of ≤5×10⁻⁶, an isotopic abundance difference of ≤5%, and a secondary spectral library matching score of ≥70%. This approach enabled the rapid screening of 1091 pesticides and their metabolites in edible fungi without the need for reference standards. The rapid screening of 1 091 pesticides and metabolites in edible fungi could be realized. The methodological validation by using 16 representative pesticides showed that the platform’s limits of detection (LODs) ranged from 2 to 1000 ng·mL⁻¹. This method had the advantages of covering a wide range of pesticides, simple pretreatment procedures, freedom from reference standard dependence, as well as high resolution and high throughput, which was suitable for the routine broad-spectrum screening of pesticide residues in edible fungi, and could provide robust technical support for food safety supervision and risk assessment.