Ginsenosides are glycosylated dammarene-type triterpenes that have been identified in distantly related Panax ginseng and Gynostemma pentaphyllum. The phylogenetic relatedness of the ginsenoside biosynthetic genes in the two species was previously unknown. The final steps of ginsenoside biosynthesis are the glycosylations of hydroxylated triterpenes, protopanaxadiol (PPD) and protopanaxatriol (PPT), and their glycosylated forms by UDPglycosyltransferases (UGTs). Ginsenoside biosynthetic UGTs have been identified in Panax but not in Gynostemma. Through a biochemical screening of Gynostemma UGTs (GpUGTs), we herein identified three groups of ginsenoside biosynthetic GpUGTs. These groups comprise: two GpUGTs that belong to the UGT71 family and glucosylate the C20-OH positions of PPD- and PPT-type ginsenosides; one GpUGT that belongs to the UGT74 family and glucosylates the C3-OH position of PPD-type ginsenosides; and two GpUGTs that belong to the UGT94 family and add a glucose to the C3-O-glucosides of PPD-type ginsenosides. These GpUGTs belong to the same UGT families as the ginsenoside biosynthetic Panax UGTs (PgUGTs). However, GpUGTs and PgUGTs belong to different subfamilies. Furthermore, cucumber UGTs orthologous to GpUGTs do not glucosylate ginsenosides. These results collectively suggest that, during evolution, P. ginseng and G. pentaphyllum independently opted to use the same UGT families to synthesize ginsenosides.