Background: Flavonoids have a wide range of biological activities in plant development, stress resistance and human health, etc. R2R3-MYBs are one of the key elements in regulation of flavonoid production, but their functional importance in Betula platyphylla remains elusive.Methods: The full-length transcriptome data of 30-day-old seedlings of Betula platyphylla were used to identify BpR2R3-MYB family genes, and their gene structure, chromosome distribution and syntenic relationships were predicted by bioinformatics methods. Agrobacterium-mediated transient transformation was used to verify the function of BpR2R3-pMYB15 in flavonoid production.Results: 44 BpR2R3-MYB family genes expressed in seedlings of Betula platyphylla were identified and found to be unevenly distributed in 11 chromosomes. Among them, 90.90% of the BpR2R3-MYBs had introns, and only four genes had no introns. Five gene pairs with segment duplication were found, and their Ka/Ks ratios were less than 1. Thirty orthologs between Betula platyphylla and Arabidopsis thaliana and 68 orthologs between Betula platyphylla and Populus trichocarpa were detected. Five BpR2R3-MYBs were clustered with R2R3-MYB genes related to flavonoid synthesis, and BpR2R3-pMYB15 had the highest correlation coefficients between the value of gene expression and flavonoid content. BpR2R3-pMYB15 was cloned, and its transient overexpression obtained using Agrobacterium-mediated transformation positively regulated flavonoid production.Conclusion: This work enriches the collection of R2R3-MYBs related to flavonoid production in seedlings of Betula platyphylla.