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Vitis amurensis grape cultivars and hybrids are mainly used to make wines in Northeast Asia with a cold winter. Anthocyanidin glucosylation at 5-O position catalyzed by 5-O-glucosyltransferase (5GT) in grape skins is crucial for color stability of red wines. To study 5GT functions in anthocyanidin diglucosides synthesis of V. amurensis, 20 5GTs were preliminarily identified from a genome-wide characterization of the UDP-glycosyltransferase family according to the reported 5GTs, which were also performed phylogenetic and bioinformatics analysis. Two important 5GTs, Vitvi0900582.t01 and Vitvi05g01269.t01, were screened through analyses of anthocyanidin diglucosides accumulation and gene expression in berry skins of three representative grape cultivars with significant differences in anthocyanin glycosylation. Fourteen alleles of each of the 5GTs were cloned from 14 V. amurensis cultivars and hybrids as well as from V. vinifera ‘Cabernet Sauvignon’, and sequence analysis and functional prediction were performed. From three perspectives of phenotype, transcriptional level, and genotype, it has been found that the functional allele at the Vitvi0900582.t01 locus of Chr 9 played a decisive role in the synthesis of abundant anthocyanidin diglucosides in V. amurensis. In addition, the trace anthocyanidin diglucosides detected in V. vinifera ‘Cabernet Sauvignon’ were led by the functional allele at the Vitvi05g01269.t01 locus of Chr 5. This study provides preliminary data for further research on the regulatory mechanism of anthocyanidin diglucosides in the grapes with the V. amurensis pedigree to improve their wine quality in future breeding efforts.