Objective To determine the contents of seven main flavonoids in Epimedium of different places of origin by high performance liquid chromatography (HPLC). Methods The HPLC condition was as follows: SHISEIDO MG-C₁₈ column (3.0 mm×100 mm,3.0 μm); the mobile phase consisted of acetonitrile and water containing 0.1% formic acid was used as gradient elute; the gradient of acetonitrile was 25% (0-10 min), 25%-40% (10-12 min), 40%-45% (12-22 min), 45%-75% (22-25 min) and 75%(25-30 min); flow rate was 0.6 mL/min; UV detection wavelength was set at 270 nm; column temperature was 25℃; and injection volume was 5 μL. The sample was ultrasonically extracted with 70% ethanol solution. Results The seven flavonoids, including epimedin A, epimedin B, epimedin C, icariin, icariside Ⅰ, icariside Ⅱ and anhydroicaritin were separated at baseline within 30 min. The standard curves had good linearity (r=0.999 9), with the RSD% of intraday and interday precision being both less than 2.0%, the recovery being 98%-102%, and the RSD% of stability and repeatability also being less than 2.0%. The results showed that the method could meet the requirements of method validation. The contents of seven flavonoids in reference crude meterial and Herba Epimedii from Liaoning, Gansu and Hubei province were determined. Conclusion The present method is rapid and simple, and can be used for quality control of Epimedium, which may lay a foundation for studying the pharmacokinetics and tissue distribution of the flavonoids of Epimedium.