Abstract: A high-performance liquid chromatography (HPLC) method was developed for simultaneous determination of quercetin and kaempferol in Melaleuca alternifolia (Maiden et Betehe) Chee 1.The sample was extractd by HCl-MeOH solution (volume ratio 5:100) in water bath, then separated by a C18 column with CH3OH-C2H3N-0.4% phosphoric acid solution (volume ratio 21:21:58) as the mobile phase solution under 370 nm wavelength. The HPLC experiment was performed at room temperature with flow rate 1.0mL/min. Quercetin and kaempferol contents in M.alternifolia from different area were determined. The result showed that quercetin and kaempferol content in Nanxiao town of Yongning was the highest and that in Yongning was the lowest.