Abstract: In this study, we first investigate and clone the R2R3−MYB genes involved in regulate of the natural rubber pathway in Periploca sepium, providing useful genes for the regulation of natural rubber biosynthesis. Using the method of bioinformatics, 122 R2R3−MYB genes were found by analyzing the genomic data of P. sepium. Through the construction of phylogenetic tree, amino acid multi sequence alignment, conserved domain and protein tertiary structure analysis, the R2R3−MYB gene that may be involved in the regulation of natural rubber synthesis was determined, and the tissue-specific expression of the gene was analyzed. The results showed that PsMYB79, PsMYB102, PsMYB159 and PsMYB178 belongs in the same evolutionary branch with HbMYB, that controlling natural rubber, and alignment of the protein sequence of these genes revealed a highly conserved R2R3 repeat domain and SHAQKYF conserved motif. The amino acid sequence similarity between these genes and HbMYB gene was 72.5%, 64.5%, 61.6% and 57.8% respectively. RT−PCR analysis showed that the 4 genes were expressed in the site of latex formation, among which PsMYB79 and PsMYB178 were the strongest in roots, PsMYB102 was strongly expressed in young leaves, and PsMYB159 was generally expressed in roots, stems, young leaves and old leaves. Therefore, it is speculated that PsMYB79, PsMYB102, PsMYB159 and PsMYB178 genes in the R2R3−MYB transcription factor family of P. sepium may be involved in the regulation of natural rubber biosynthesis in different tissues and organs of plants.