MYB family of transcription factors (TF) comprises one of the largest transcription factors in plants and is represented in all eukaryotes. They include highly conserved MYB repeats (1R, R2R3, 3R, and 4R) in the N-terminus. In addition to this, they have diverse C-terminal sequences which help the protein gain wide distinct functions, such as controlling development, secondary metabolism, hormonal regulation and response to biotic and abiotic stress. Stress-responsive roles of the MYB TFs were reported for drought, salt, wounding, cold, freezing, dehydration and osmotic stresses. This study describes the identification of barley R2R3-MYB TFs including their expression analysis in tissues under control and Boron (B) toxic conditions. Conserved motifs for MYB proteins were searched into barley full-transcriptome RNA-seq data and a total of 320 protein sequences were filtered as MYB TFs in which 51 of them corresponded to R2R3 MYB TFs. Using various bioinformatics tools, their conserved domain structures, chromosomal distributions, gene duplications, comparative functional analysis, as well as phylogenetic relations with Arabidopsis thaliana, were conducted. Beside the RNA-seq data-based expression pattern analysis of 51 R2R3 MYB TFs, quantitative analysis of selected R2R3 MYB TF genes was assessed in control and B-stressed root and leaf tissues. Critical B-induced R2R3 MYB TFs were identified. It was concluded that the results would be useful for functional characterizations of R2R3-type MYB transcription factors that are possibly involved in both B stress and divergent regulation mechanisms in plants.