High-performance liquid chromatographic coupled with variable wavelength detection (HPLC-VWD) has been developed for simultaneous determination of 5 analytes including ellagic acid, quercetin, kaempferol-3-O-beta-D-rutinoside, tiliroside and kaempferol, and high-performance liquid chromatographic with an evaporative light scattering detector (HPLC-ELSD) has been established to determine goshonoside-F5 in extract of Rubi Fructus. Chromatographic separations were carried out on an Agilent ZORBAX SB-C18 column (4.6 mm x 250 mm, 5.0 microm). All calibration curves of reference standards revealed good linearity (R2 > 0.999 5) within the concentration ranges tested. The method limits of detection ranged 0.297-90.144 ng and the method limits ofquantitation ranged 0.990-300.480 ng, respectively. Recoveries of 6 analytes were from 97.11% to 101.7%, with RSD less than 2.1%. The result shows that amounts of the 6 analytes in the samples from 16 localities were found to be different. The higher latitude of growing environment, the more ellagic acid in herb. The content of total flavonoids in sample from east localities were higher than that in middle and west localities, and the content of goshonoside-F5 in Bozhou, Anhui province was higher than others. This method was found to be simple, accurate, sensitive with good repeatability. Those results might serve as a sound foundation for further study, quality control and application of Rubi Fructus.