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R2R3-MYB transcription factors (TFs) form one of the most important TF families involved in regulating various physiological functions in plants. The heartwood of <i>Dalbergia odorifera</i> is a kind of high-grade mahogany and valuable herbal medicine with wide application. However, the role of R2R3-MYB genes in the growth and development of <i>D. odorifera</i>, especially their relevance to heartwood formation, has not been revealed. A total of 126 R2R3-MYBs were screened from the <i>D. odorifera</i> genome and named DodMYB1-126 based on their location on 10 chromosomes. The collinearity results showed that purification selection was the main driving force for the evolution of the R2R3-MYB TFs family, and whole genome/fragment replication event was the main form for expanding the R2R3-MYB family, generating a divergence of gene structure and function. Comparative phylogenetic analysis classified the R2R3-MYB TFs into 33 subfamilies. S3-7,10,12-13,21 and N4-7 were extensively involved in the metabolic process; S9,13,16-19,24-25 and N1-3,8 were associated with the growth and development of <i>D. odorifera</i>. Based on the differential transcriptional expression levels of R2R3-MYBs in different tissues, DodMYB32, DodMYB55, and DodMYB89 were tentatively screened for involvement in the regulatory process of heartwood. Further studies have shown that the DodMYB89, localized in the nucleus, has transcriptional activation activity and is involved in regulating the biosynthesis of the secondary metabolites of heartwood by activating the promoters of the structural genes <i>DodI2’H</i> and <i>DodCOMT</i>. This study aimed to comprehensively analyze the functions of the R2R3-MYB TFs and screen for candidate genes that might be involved in heartwood formation of <i>D. odorifera</i>.