Objective To establish the HPLC fingerprint and pesticide residue detection methods for different kinds of rhubarb, and evaluate the quality of rhubarb comprehensively. Methods 20 batches of three types of rhubarb were collected and analyzed by high-performance liquid chromatography. The mobile phase was methanol-0.1 % phosphoric acid solution; gradient elution; column temperature of 35 ℃; detection wavelength of 254 nm; flow rate 1.0 ml/min. And cluster analysis was performed on the results. Direct extraction method was used and high-performance liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry were established, 33 prohibited pesticides from different sources and origins of rhubarb were detected. Results The similarity among the fingerprint spectra of three sources of rhubarb, namely Rheum palmatum L., Rheum tanguticum Maxim.ex Balf., and Rheum officinale Bail1., and their control fingerprint spectra was>0.95. 20 batches of rhubarb samples were divided into 3 categories by cluster analysis. 33 prohibited pesticides were detected in rhubarb samples from different regions. Conclusion The quality of three kinds of rhubarb was significantly different. The established HPLC fingerprint and the method of banning agricultural residues were stable, reliable, simple and accurate, which could provide a basis for quality control evaluation of rhubarb.