Kudzu () is used as a traditional medicine in China and Southeast Asia but is a noxious weed in the Southeastern United States. It produces both - and glycosylated isoflavones, with puerarin (-glucosyl daidzein) as an important bioactive compound. Currently, the stage of the isoflavone pathway at which the glycosyl unit is added remains unclear, with a recent report of direct glycosylation of daidzein contradicting earlier labeling studies supporting glycosylation at the level of chalcone. We have employed comparative mRNA sequencing of the roots from two species, one of which produces puerarin (field collected ) and one of which does not (commercial ), to identify candidate uridine diphosphate glycosyltransferase (UGT) enzymes involved in puerarin biosynthesis. Expression of recombinant UGTs in and candidate -glycosyltransferases in were used to explore substrate specificities, and gene silencing of UGT and key isoflavone biosynthetic genes in kudzu hairy roots employed to test hypotheses concerning the substrate(s) for glycosylation. Our results confirm UGT71T5 as a -glycosyltransferase of isoflavone biosynthesis in kudzu. Enzymatic, isotope labeling, and genetic analyses suggest that puerarin arises both from the direct action of UGT71T5 on daidzein and via a second route in which the C-glycosidic linkage is introduced to the chalcone isoliquiritigenin.