Abstract Background R2R3-MYB transcription factors regulate secondary metabolism, stress responses and development in various plants. Puerarin is a bioactive ingredient and most abundant secondary metabolite isolated from Pueraria lobata . The biosynthesis of puerarin proceeds via the phenylpropanoid pathway and isoflavonoids pathway, in which 9 key enzymes are involved. The expression of these structural genes is under control of specific PtR2R3-MYB genes in different plant tissues. However, how PtR2R3-MYB genes regulates structural genes in puerarin biosynthesis remains elusive. This study mined the PtR2R3-MYB genes involved in puerarin biosynthesis and response to hormone in Pueraria lobata var. thomsonii . Results A total of 209 PtR2R3-MYB proteins were identified, in which classified into 34 subgroups based on the phylogenetic topology and the classification of the R2R3-MYB superfamily in Arabidopsis thaliana . Furtherly physical and chemical characteristics, gene structure, and conserved motif analysis were also used to further analyze PtR2R3-MYBs . Combining puerarin content and RNA-seq data, speculated on the regulated puerarin biosynthesis of PtR2R3-MYB genes and structural genes, thus 21 PtR2R3-MYB genes and 25 structural genes were selected for validation gene expression and further explore its response to MeJA and GSH treatment by using qRT-PCR analysis technique. Correlation analysis and cis-acting element analysis revealed that 6 PtR2R3-MYB genes ( PtMYB039 , PtMYB057 , PtMYB080 , PtMYB109 , PtMYB115 and PtMYB138 ) and 7 structural genes ( PtHID2 , PtHID9 , PtIFS3 , PtUGT069 , PtUGT188 , PtUGT286 and PtUGT297 ) were directly or indirectly regulation of puerarin biosynthesis in ZG11. It is worth noting that after MeJA and GSH treatment for 12–24 h, the expression changes of most candidate genes were consistent with the correlation of puerarin biosynthesis, which also shows that MeJA and GSH have the potential to mediate puerarin biosynthesis by regulating gene expression in ZG11. Conclusions Overall, this study provides a comprehensive understanding of the PtR2R3-MYB and will paves the way to reveal the transcriptional regulation of puerarin biosynthesis and response to phytohormone of PtR2R3-MYB genes in Pueraria lobata var. thomsonii .