Background: R2R3-MYB genes comprise one of the largest and most important gene families in plants, and are involved in the regulation of plant growth and development as well as responses to abiotic stresses. However, the functions of R2R3-MYB genes in Dendrobium nobile remains largely unknown. Methods: Here, a comprehensive genome-wide analysis of D. nobile R2R3-MYB genes was performed, in which phylogenic relationships, gene structures, motif composition, chromosomal locations, collinearity analysis, and cis-acting elements were investigated. Moreover, the expression patterns of selected DnMYB genes were analyzed in various tissues and under different abiotic stresses. Results: In total, 125 DnMYB genes were identified in the D. nobile genome, and were subdivided into 26 groups based on phylogenetic analysis. Most genes in the same subgroup showed similar exon/intron structure and motif composition. All the DnMYB genes were mapped to 19 chromosomes with the co-linearity relationship. Reverse transcription-quantitative real-time PCR (RT-qPCR) results showed that 8 DnMYBs exhibited different expression patterns in different plant tissues, and were differentially expressed in response to abscisic acid, methyl jasmonate, low-temperature stress. Conclusions: This work contributes to a comprehensive understanding of the R2R3-MYB gene family in D. nobile, and provides candidate genes for future research on abiotic stress in this plant.