Abstract: A new analytical method for the simultaneous determination of threeconstituents, viz, mangiferin, isomangiferin and chlorogenic acid in Folium Pyrrosiaeusing high performance liquid chromatography was developed. A Zorbax-CN co-lunm (4.6mm×25cm) was used; methanol- water- phosphoric acid (pH 2.5) (20:80: 0.1) was used as the mobile phase. The method was rapid, sensitive, precise andshowed good reproducibility. The three constituents in crude drugs were completely sepa-rated within 15 min. Recoveries of three constituents were 96,78～101.3%, withcoefficients of variation 1.8 ～4.7%. Mangiferin and isomangiferin were separated for thefirst time. Pyrrosia plant materials were analyzed as follows: The powdered sample was refluxed with methanol for 4 hours, the extract wastransferred into a 25 mi volumetric flask and diluted to 25 ml with methanol. A definiteamount of this sample solution was injected for HPLC. The content of constituents inFolium pyrrosiae was calculated from the relevant peak height or peak area. The content of these three constituents in 7 species of Chinese Pyrrosia collected in 17districts were determined. The results showed that these constituents varied greatly withthe plant species and varied with the districts for the same species also, and should be eva-luated accordingly.