OBJECTIVE:To study HPLC fingerprints of Arnebiae Radix,and to determine the contents of beta-acetoxyisovaleryalkannin. METHODS:The determination was performed on ACE C18 column(250 mm*4.6 mm,5 mum)with mobile phase consisted of acetonitrile-0.1 % formic acid(70 ∶ 30,V/V)at the flow rate of 1.0 ml/min. The detection wavelength was set at 516 nm. RESULTS: HPLC fingerprints of Arnebiae Radix showed good separation,precision,reproducibility and stability;the similarity of the fingerprints in 10 batches of Arnebiae Radix was more than 0.950. The linear range of beta-acetoxyisovaleryalkannin were 0.3-9.0 mug(r=0.999 9)with an average recovery of 101.40%(RSD=4.08%,n=6). The contents of beta-acetoxyisovaleryalkannin from different habitats were significantly different from each other. CONCLUSIONS:beta-acetoxyisovaleryalkannin can be good chemical marker for the content determination and fingerprint study of Arnebiae Radix. It can provide basis for the verification standards of Arnebiae Radix in Chinese Pharmacopeia(2015 edition).