Abstract: A new and rapid analytical method for the simultaneous determination of five bitter secoiridoid glycosides( gentiopicroside, GTP; swertiamarin, SWT; sweroside, SWO; amarogentin, AMG; amaroswerin, AMS)in the Chinese drug "Long Dan", roots of Gentiana manshurica and 8 allied species by high performance liquid chromatography (HPLC)has been developed. The HPLC system consisting of an apparatus from Shimadzu (model LC-4A), with a UV-detector(SPD-2AS), a data processor (Chromatopac C-R2AX), a column oven (CTO- 2AS), and a Zorbax ODS column (25 cm×4.6 mm ID) was used. Using MeOH-H₂O as the mobile phase, the linear concentration-program of methanol was time (min) : C (MeOH) : 0:20→6 : 20→ 6.01: 401→23:stop. The temperature of column oven was 40℃. The bitter secoiridoid glycosides eluted were detected at a wavelength of 254 nm and the analysis was successfully carried out within 23 minutes. This method is sensitive, rapid, accurate and has good reproducibility. Recoveries of each secoiridoid glycoside were 100.0～101.5% with coefficients of variation 0～2.5% (n=3). The contents of five bitter secoiridoid glycosides in the roots of G. manshurica and eight allied species indigenous to China were determined and reported.