Objective:To determine paeonol in Cynanchum paniculatum from different habitats by HPLC to provide better quality evaluation for Cynanchum paniculatum. Methods:A Promosil C18 column (200 mm × 4. 6 mm, 5 μm) was adopted, methanol-water (55∶45) was used as the mobile phase, the flow rate was 1. 0 ml·min-1 , the detection wavelength was at 274 nm, and the injection volume was 10μl. Results:Paeonol showed a good linearity(r=0. 999 9) within the range of 0. 049 4-0. 790 8 mg·ml-1,and the re-covery was 99. 98% with RSD of 0. 17%. The content of paeonol in Cynanchum paniculatum from different habitats showed significant difference. Conclusion:The method is simple, accurate and reproducible, which can be used to determine the content of paeonol in Cynanchum paniculatum from different habitats.