In this study, the biosynthesis pathway and regulatory mechanism of triterpenoids in Rosa roxburghii (R. roxburghii) were systematically investigated through combined use of liquid chromatography-mass spectrometry and transcriptome analysis. The results showed that there were significant differences in triterpenoid content between cultivated and wild R. roxburghii varieties, with the cultivated variety exhibiting an approximately 30 wt.% higher triterpenoid content compared with the wild variety. Such a difference remained constant in the different growth stages of R roxburghii. Through high-throughput transcriptome sequencing technology, five key genes associated closely with the biosynthesis of triterpenoid were successfully identified in this research. These genes had high homology to genes such as CYP716A1, CPY734A1, CPY72A15, UGT74F2, and UGT85A2, suggesting their key roles in the synthesis of triterpenoid in R. roxburghii. In this study, the combined metabolomic and transcriptome analysis not only clarified the differences in the triterpenoid content of R. roxburghii and also successfully identified the key genes participating in the biosynthesis of triterpenoids, which offers fresh insights and potential molecular markers for the quality improvement and molecular breeding of R. roxburghii.