Objective: To establish the HPLC fingerprints of Ophioglossum pedunculosum

O.vulgatum and O.thermal of O. and provide basis for its quality evaluation. Method: HPLC method was used on Waters Xbridget column (4.6 mm×250 mm

5 μm) with the mobile phase of methanol (A)-0.3%methanoic acid solution (B) for gradient elution. The flow rate was 1.0 mL·min-1and the column temperature was 40 ℃. The detection wavelength was set at 260 nm

and the injection volume was 10 μL. The fingerprints of Ophioglossum were analyzed by cluster analysis

similarity comparative analysis and principal component analysis. Result: Fifteen batches of Ophioglossum were classified into three kinds

and 14 main common peaks were identified in the fingerprints. The genetic relationship of O.vulgatum and O.thermal was close to each other

while far away from O.pedunculosum. Four control substances including ophioglonol

3-O-methylquercetin

ophioglonol4'-O-β-D-glucopyranoside

and 3-O-methylquercetin7-O-β-D-glucopyranosyl- 4'-O-β-D-glucopyranoside can be served as chemotaxonomy tags for distinguishing the above three kinds. Conclusion: The establishment of HPLC fingerprint of Ophioglossum provides a reference method for quality control and species identification of its original plants.