MYB transcription factors (TFs) make up one of the most important TF families in plants. These proteins play crucial roles in processes related to development, metabolism, and stimulus-response; however, very few studies have been reported for the characterization of MYB TFs from banana. The current study identified 305 and 251 MYB genes from Musa acuminata and Musa balbisiana, respectively. Comprehensive details of MYBs are reported in terms of gene structure, protein domain, chromosomal localization, phylogeny, and expression patterns. Based on the exon-intron arrangement, these genes were classified into 12 gene models. Phylogenetic analysis of MYBs involving both species of banana, Oryza sativa, and Arabidopsis thaliana distributed these genes into 27 subfamilies. This highlighted not only the conservation, but also the gain/loss of MYBs in banana. Such genes are important candidates for future functional investigations. The MYB genes in both species exhibited a random distribution on chromosomes with variable densities. Estimation of gene duplication events revealed that segmental duplications represented the major factor behind MYB gene family expansion in banana. Expression profiles of MYB genes were also explored for their potential involvement in acetylene response or development. Collectively, the current comprehensive analysis of MYB genes in both species of banana will facilitate future functional studies.