Background Studies on pyrrolizidine alkaloid(PA) contamination in honey produced in China are scarce. Previously reported HPLC-MS/MS methods for the determination of PAs in honey often suffer from insufficient separation and uncertainties of PA isomers. Objective To develop and validate an UHPLC-MS/MS method for base-line separation of PA isomers towards precise determination of 32 PAs in Chinese wild honey. Method PAs were extracted from honey samples and separated on an ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm) column with (A) 0.1% formic acid aqueous solution containing 5 mM ammonium acetate and (B) methanol as mobile phase. The column temperature was maintained at 30 °C, and flow rate was 0.3 mL/min. Detection was performed by tandem mass spectrometry. The total runtime was reduced to 18 min. Results 31 of 32 PAs were baseline separated efficiently within 18 min. The limits of detection(LOD) and quantification(LOQ) were 0.06–0.25 μg/kg and 0.22–0.82 μg/kg, respectively, except for that of clivorine, for which LOD and LOQ were 2.03 and 6.78 μg/kg, respectively. The average recoveries ranged between 66.3% and 95.1% and the average relative standard deviations (RSD) were 3.2% to 8.0%. The established method was used to analyze PAs in 22 types of Chinese wild honey, and the predominant PAs found in these honey samples were intermedine and lycopsamine. Conclusions A high-throughput method for the determination of isomeric PAs in honey was developed and validated. And 5 of the 22 types of Chinese wild honey were contaminated with PAs concentrations of 2.2–207.0 μg/kg. Highlights A new method capable of monitoring more PAs and to provide better separation than previously reported protocols for the determination of multiclass PAs in honey is established.