To establish a mass spectrometry method for the determination of characteristic peptides of Hirudo from different origins,and to provide a basis for the identification and quality evaluation.The Agilent ZORBAX SB C₁₈ column (100 mm×2.1 mm,1.8 μm) was used and eluted with acetonitrile (A) and 0.1% formic acid solution (B) in gradient mode at a flow rate of 0.3 mL/min.The column temperature was 30 ℃.The injection volume was 2 μL.The samples were extracted and enzymolysis treatment used trypsin.The characteristic molecular peaks of Hirudo m/z 601 (double-charge) → 630.35 and m/z 601 (double-charge) → 743.44,m/z 475 (triple-charge) →588.76 and m/z 475 (triple-charge) → 662.30 were detected by UPLC-MS/MS.The results showed that Whitmania pigra Whitman can detect Hirudo polypeptide Ⅰ and Hirudo polypeptide Ⅱ,Whitmania acranulata Whitman can only detect Hirudo polypeptide Ⅱ,Hirudo nipponica Whitman can only detect Hirudo polypeptide Ⅰ,so the method can distinguish Hirudo from different origins.The SIMCA-p 14.0 software were used to analyze 22 batches of samples,the partial least squares discriminant analysis (PLS-DA) divided Whitmania pigra Whitman,Whitmania acranulata Whitman and Hirudo nipponica Whitman into three groups.The method established in this study was specific,precise and reproducible,which can be used to distinguish Hirudo from different origins,improve the quality control standard,and provide a reference for the safety and effectiveness of Hirudo in clinical medicine.