For the first time, a novel, simple and reliable method for analysis of pymetrozine residues in flue-cured tobacco leaves has been developed utilizing HPLC-UV with liquid-liquid partition cleanup. Pre-treatment with ultrasonic extraction and liquid-liquid partition procedures gave preferable baseline separation and clean chromatograms by removing water-soluble and fat-soluble components which interfere with pymetrozine in the test. The performance of the method was evaluated and validated: the detection limit (LOD) was 0.005 microg x mL(-1), the relative standard deviation (RSD) was 1.2% (n = 5), and the overall recovery was above 90% at fortification levels of 0.200, 0.500, 1.000, and 5.000 mg x kg(-1). The proposed method was successfully employed for the determination of pymetrozine residues in twelve flue-cured tobacco samples collected from different regions of China.