Abstract: In order to determine the chemical species of arsenic (As) in tobacco samples, a method combining ultrasonic solvent extraction and high performance liquid chromatography (HPLC) with inductively coupled plasma-mass spectrometry (ICP-MS) was developed to separate and analyze six species of arsenic, including arsenous acidAs(Ⅲ), arsenic acidAs(Ⅴ), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), arsenobetaine (AsB), and arsenocholine (AsC). The result showed that the baselines of the six species of arsenic mentioned above were separated within 15 mins by Hamilton PRPX-100(250×4.1 mm,10μm) anion exchange column and using 15 mmol/L (NH₄)₂HPO₄ (pH6.0) as the mobile phase. The linear ranges were no less than two orders of magnitude. The detection limit ranged from 0.44 to 0.98μg/L and the recoveries were from 85.19% to 87.31% with the RSD (n=5) of 2.45% to 3.56%. The content of As in tobacco was low, and some arsenic species existed in the forms of DMA and MMA were of lower toxicity. Ultrasonic solvent extraction could extract the organic and inorganic As in tobacco simultaneously and reflect the distribution of As species in tobacco samples objectively. This method is accurate, reliable, and suitable for the speciation analysis of As in tobacco, and can provide a reference for the safety evaluation of tobacco.