Betanidin 6-O-glucosyltransferase (6-GT) is involved in the glycosylation of betacyanins, which replace the chromogenic anthocyanins as flower colorants in the Caryophyllales. The 6-GT cDNA was cloned from a cDNA library of Dorotheanthus bellidiformis (Burm. f.) N.E. Br., and the amino acid and nucleotide sequences were shown to be distinctly different from the corresponding betanidin 5-O-glucosyltransferase (5-GT) from the same plant species. Although both enzymes share very similar substrates, the proteins show only 19% amino acid sequence identity. In contrast, the protein sequence of the 6-GT showed significant identity to GTs from other species and may identify a new cluster of putative anthocyanidin GTs. Therefore, 6-GT and 5-GT apparently have evolved independently from ancestral glucosyltransferases involved in flavonoid biosynthesis.