Abstract: A method for HPLC fingerprinting of the aerial parts of Gentiana rigescens Franch. ex Hemsl. in agroforestry and monoculture systems was established. Contents of the main bioactive compounds, such as loganic acid, swertiamarin, gentiopicroside and sweroside, were determined. Variations in the chemometric characteristics of the stems and leaves under different cultivation systems were investigated, with correlation analysis, similarity analysis, partial least squares discriminant analysis (PLS-DA) and variable importance in the projection (VIP) analysis used to investigate these variations. Quantitative analysis showed that gentiopicroside, swertiamarin, loganic acid and sweroside contents ranged from (15.28±11.34) mg/g to (24.59±7.84) mg/g, (4.10±1.64) mg/g to (31.67±22.70) mg/g, (1.85±0.92) mg/g to (7.43±7.64) mg/g, and (1.03±0.17) mg/g to (1.58±0.50) mg/g, respectively. Total contents of the four bioactive compounds in leaves were higher than those in stems. Total content of the four bioactive compounds in the aerial part was the highest in the plant intercropped with Alnus nepalensis D. Don and was the lowest in the plant intercropped with Juglans regia L.. Correlation analysis showed that the variation in iridoid and secoiridoid content had significant (P < 0.05) or very significant (P < 0.01) positive correlation in the same part and in different parts, respectively. Similarities of the stem samples ranged from 0.989 to 0.992. Similarities of the leaf samples ranged from 0.988 to 0.996. The chemical constituents in stems and leaves were similar. However, the contents of those compounds were different. PLS-DA analysis showed that the chemometric characteristics of the stems and leaves were very different. Samples collected from different cultivation systems were classified as different groups. Chemical compounds in the leaves were more affected by the cultivation system than those in the stems. These results provide useful information for the study of agroforestry systems of G. rigescens Franch. ex Hemsl. and sustainable use of medicinal materials.