###### Abstract  
_Gynostemma pentaphyllum_ is a traditional Chinese medicinal plant of considerable application value and commercial potential, primarily due to its production of various bioactive compounds, particularly dammarane-type triterpenoid saponins that are structurally analogous to ginseng. Oxidosqualene cyclase (OSC), a pivotal enzyme in the biosynthesis of triterpen metabolites in plants, catalyzes the conversion of oxidosqualene into triterpenoid precursors, which are essential components of the secondary metabolites found in _G. pentaphyllum_. To elucidate the role of _OSC_ gene family members in the synthesis of gyneposides within _G. pentaphyllum_, this study undertook a comprehensive genome-wide identification and characterization of _OSC_ genes within _G. pentaphyllum_ and compared their expression levels across populations distributed over different geographical regions by both transcriptome sequencing and qRT-PCR experimental validation. The results identified a total of 11 members of the _OSC_ gene family within the genome of _G. pentaphyllum_. These genes encode proteins ranging from 356 to 767 amino acids, exhibiting minor variations in their physicochemical properties, and are localized in peroxisomes, cytoplasm, plasma membranes, and lysosomes. All _GpOSCs_ contain highly conserved DCTAE and QW sequences that are characteristic of the _OSC_ gene family. A phylogenetic analysis categorized the _GpOSCs_ into four distinct subfamilies. A cis-element analysis of the _GpOSC_ promoters revealed a substantial number of abiotic stress-related elements, indicating that these genes may respond to drought conditions, low temperatures, and anaerobic environments, thus potentially contributing to the stress resistance observed in _G. pentaphyllum_. Expression analyses across different _G. pentaphyllum_ populations demonstrated significant variability in _OSC_ gene expression among geographically diverse samples of _G. pentaphyllum_, likely attributable to genetic variation or external factors such as environmental conditions and soil composition. These differences may lead to the synthesis of various types of gyneposides within geographically distinct _G. pentaphyllum_ populations. The findings from this study enhance our understanding of both the evolutionary history of the _OSC_ gene family in _G. pentaphyllum_ and the biosynthetic mechanisms underlying triterpenoid compounds. This knowledge is essential for investigating molecular mechanisms involved in forming dammarane-type triterpenoid saponins as well as comprehending geographical variations within _G. pentaphyllum_ populations. Furthermore, this research lays a foundation for employing plant genetic engineering techniques aimed at increasing gypenoside content. Gynostemma pentaphyllum; triterpene saponins; 23-oxidized squalene cyclase; _OSC_ gene family; gene expression 202 in tropical Asia, particularly in the southern regions of the Qinling Mountains and the moist forests of the Yangtze River Basin in China [1]. As a traditional Chinese medicinal plant, _G. pentaphyllum_ is commonly referred to as \"Southern ginseng\" and possesses significant medicinal value due to its content of ginsenosides and other structurally similar dammarane-type saponins [2]. Furthermore, _G. pentaphyllum_ contains various bioactive compounds including saponins, flavonoids, amino acids, and vitamins [3]. Among these constituents, gypenosides are recognized as the primary active ingredients, which exhibit antioxidant properties along with anti-cancer effects, lipid-lowering capabilities, and immune-enhancing activities [4; 5; 6]. Currently, _G. pentaphyllum_ has been extensively developed into health care products such as oral liquids, health capsules, and teas, which present vast commercial potential [7]. In recent years, there has been growing interest in developing drugs and health foods containing gypenosides; this trend presents both unprecedented opportunities and challenges for harnessing _G. pentaphyllum_ resources.