Swertia chirayita is an endangered medicinal herb widely used as an antidiabetic. It contains two major classes of metabolites, secoiridoids and xanthones, i.e. swertiamarin, mangiferin, amarogentin and amaroswerin. The biosynthetic pathways for these chemical constituents are not completely deciphered due to gaps and redundancy of routes proposed such as for mangiferin. The missing intermediates in pathways were detected through LC-ESI-QToF-HRMS/MS, including the detection of new secoiridoids, amaronitidin and gentiopicroside. The study also reports that the biosynthesis of amaronitidin occurs through the coupling of gentiopicroside and biphenyl acid derivatives such as amarogentin and amaroswerin. This study reports for the first time complete biosynthetic pathways for gentiopicroside, mangiferin, amarogentin, amaroswerin and amaronitidin in S. chirayita with the detection of intermediate metabolites iriflophenone, maclurin, deoxyloganic acid, loganic acid and 1,3,6,7-tetrahydroxy-9H-xanthen-9-one.