The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wide analyses of this gene family have been conducted in several species, R2R3-MYB genes have not been systematically analyzed in Medicago truncatula, a sequenced model legume plant. Here, we performed a comprehensive, genome-wide computational analysis of the structural characteristics, phylogeny, functions and expression patterns of M. truncatula R2R3-MYB genes. DNA binding domains are highly conserved among the 155 putative Mt R2R3-MYB proteins that we identified. Chromosomal location analysis revealed that these genes were distributed across all eight chromosomes. Results showed that the expansion of the Mt R2R3-MYB family was mainly attributable to segmental duplication and tandem duplication. A comprehensive classification was performed based on phylogenetic analysis of the R2R3-MYB gene families in M. truncatula, Arabidopsis thaliana and other plant species. Evolutionary relationships within clades were supported by clade-specific conserved motifs outside the MYB domain. Species-specific clades have been gained or lost during evolution, resulting in functional divergence. Also, tissue-specific expression patterns were investigated. The functions of stress response-related clades were further verified by the changes in transcript levels of representative R2R3-MYB genes upon treatment with abiotic and biotic stresses. This study is the first report on identification and characterization of R2R3-MYB gene family based on the genome of M. truncatula, and will facilitate functional analysis of this gene family in the future. The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wide analyzes of this gene family have been conducted in several species, R2R3-MYB genes have not been systematically analyzed in Medicago truncatula, a sequenced model legume plant. Here, we performed a comprehensive, genome-wide computational analysis of the structural characteristics, phylogeny, functions and expression patterns of M. truncatula R2R3-MYB genes Chromosomal location analysis revealed those genes were distributed across all eight chromosomes. Results showed that the expansion of the Mt R2R3-MYB family was primarily attributable to segmental duplication and tandem duplication. A comprehensive classification was performed based on phylogenetic analysis o f the R2R3-MYB gene families in M. truncatula, Arabidopsis thaliana and other plant species. Evolutionary relationships within clades were supported by clade-specific conserved motifs outside the MYB domain. Species-specific clades have been gain or lost during evolution, resulting in functional diver stress. Also functions of stress response-related clades were further verified by the changes in transcript levels of representative R2R3-MYB genes upon treatment with abiotic and biotic stresses. This study is the first report on identification and characterization of R2R3-MYB gene family based on the genome of M. truncatula, and will facilitate functional analysis of this gene family in the future.