Eureka lemon from 3 different areas: Anyue county at Sichuan province, Ruili county of Yunnan province, Heyuan city at Guangdong province and 3 different varieties: Lisben, Mayer lemon and Rough lemon from Beibei region at Chongqing city were used as material, to establish a high performance liquid chromatograph (HPLC) method for the determination of 11 flavonoids, namely, hesperidin, neohesperidin, naringin narirutin, eriocitrin, hesperitin, naringenin, luteolin, nobiletin, eriodictyol and rutin in lemon fruits simultaneously. The results showed that 11 flavonoids were thoroughly separated within 50 min under the condition of using acetonitrile–0.1% formic acid aqueous solution as mobile phase through gradient elution and the detection wavelength was 287 nm. This method exhibited a linear range from 0.010 0 to 100.000 0 μg/mL (the correlation coefficient was from 0.999 9 to 1.000 0). The limits of detection ranged from 0.020 to 0.079 μg/mL, and the limits of quantification ranged from 0.065 to 0.263 μg/mL. The recoveries ranged from 95.49% to 103.10% with RSD between 0.81% and 1.20%. Hesperidin was the major flavonoid in six lemon varieties. Content of hesperidin, in six varieties peels was from 14.42 to 27.37 mg/g, eriocitrin was from 0.10 to 9.38 mg/g and narirutin was from 0.18 to 0.52 mg/g. Other 8 compounds in peels was less than 0.50 or not detected. Content of hesperidin in six varieties juice was from 6.64 to 7.42 mg/g, and Content of narirutin was from 0.03 to 0.15 mg/g, content of eriocitrin in juice was from 0.03 to 0.17 mg/g and not detected in Lisben and Mayer lemon, content of rutin in juice was from 0.08 to 0.13 mg/g and not detected in Lisbon from Beibei district at Chongqing, content of nobiletin was 0.05, 0.06 and 0.06 mg/g in juice of Eureka from Anyue county at Sichuan province, Lisben and Rough lemon from Beibei district at Chongqing, other 6 compounds were not detected. The results indicated that the content of flavonoids existed difference in different lemon varieties and Eureka lemon from different areas.