Mulberry is a widespread and important economic crop in China. Cutting is a suitable method for large-scale propagation of fine cultivars, but it is limited by the low rate of adventitious root (AR) formation. Currently, we found large variations of R2R3-MYB genes during AR formation in Morus alba. There are a few studies on this issue, and therefore a systematic research is needed to improve the cuttage technique. The Hidden Markov Model (HMM) was built and applied in searching MYB genes. Phylogenetic relationships with subtribe designations and clustering expression of R2R3-MYB proteins were obtained referring to the transcriptome sequencing data in the normal cutting process. In addition, hormone trials with exogenous hormone treatment were performed to observe R2R3-MYB gene expression levels by quantitative real-time PCR (qRT-PCR) along with physiological changes during AR formation. In this study, we first identified 116 R2R3-MYB family genes of 247 MYB genes in mulberry (M. alba). They are clustered into 51 subtribes according to the structures of motifs and phylogenetic classes. On the basis of data analysis, the majority of R2R3-MYB genes play roles as activating transcription factors in the cortex during AR formation, which synchronously participate in many related signal pathways. Our results showed the R2R3-MYB genes have a detailed classification and express differently in the process of cutting, indicating that this gene family plays important roles during AR formation and functions differently in response to the corresponding signal pathways.