Flavonoids are widely distributed in mulberry leaves and have been recognized for their beneficial physiological effects on the human health. Here, we analyzed variations in 44 flavonoid compounds among 91 mulberry resources. Metabolic profiling revealed that O-rhamnosylated flavonols and malonylated flavonol glycosides, including rutin and quercetin 3-O-(6″-O-malonylglucoside) (Q3MG), were absent from Morus notabilis and multiple mulberry (Morus alba L.) resources. Transcriptome and phylogenetic analyses of flavonoid-related UDP-glycosyltransferases (UGTs) suggested that the flavonol 3-O-glucoside-O-rhamnosyltransferase (FGRT) KT324624 is a key enzyme involved in rutin synthesis. A recombinant FGRT protein was able to convert kaempferol/quercetin 3-O-glucoside to kaempferol 3-O-rutinoside (K3G6″Rha) and rutin. The recombinant FGRT was able to use 3-O-glucosylated flavonols but not flavonoid aglycones or 7-O-glycosylated flavonoids as substrates. The enzyme preferentially used UDP-rhamnose as the sugar donor, indicating that it was a flavonol 3-O-glucoside: 6″-O-rhamnosyltransferase. This study provided insights into the biosynthesis of rutin in mulberry.