To cover the rising demand for natural food dyes, new sources and production methods are needed. Microbial fermentation of nature-identical colours, such as the red pigment betanin, has the potential to be a cost-efficient alternative to plant extraction. The last step of betanin production is catalysed by a UDP-glycosyltransferase (UGT). To find a high-performing UGT, we screened 27 UGTs from different plant species and tested their ability to produce betanin in vivo in Saccharomyces cerevisiae. We identified two new UGTs likely involved in the betanin synthesis in the plant they derive from: CqGT2 (UGT73A37) from Chenopodium quinoa and BgGT2 (UGT92X1) from Bougainvillea glabra. The betanin-producing UGTs were also tested in Yarrowia lipolytica, where CqGT2 was the best-performing glycosyltransferase for betanin production. While it has previously been shown that the UGTs can glycosylate either betanidin or cyclo-DOPA to ultimately form betanin, the molecular mechanism behind the preference for the acceptor molecule has not been elucidated. Therefore, we performed in silico structural analysis to characterise the betanin-producing UGTs further, particularly by looking into their binding mechanism. The docking model suggested that a smaller binding site found in some UGTs only allows glycosylation of cDOPA, while a wider binding site allows glycosylation of both cyclo-DOPA and betanidin.