Abstract: Leaf rust caused by Melampsora larici-populina is the main disease affecting poplar plantations, causing severe economic losses. To explore the regulatory effects of clade 14 R2R3-MYB genes on hypersensitive response of the poplar infected with M. larici-populina, leaves of two poplar species were inoculated by M. larici-populina E4 pathotypes to observe the symptom and score the infectious index. In addition, changes of the gene expression level of six clade 14 R2R3-MYB genes were analyzed. After seven days of inoculation, the number of uredinium on Populus deltoidesP. trichocarpa (less susceptible to M. larici-populina E4 pathotypes) leaves was significantly less than that on P.euramericana (very susceptible to M. larici-populina E4 pathotypes). On the fourth day of inoculation, symptoms of hypersensitive reaction (HR) were observed on leaves of P. deltoidesP. trichocarpa, but not on Populuseuramericana leaves. It turns out that HR can prevent the formation of uredinium and reduce the susceptibility of poplar. The change trend of R2R3-MYB gene expression level of the two poplar species was contrary. It is estimated that poplar clade 14 R2R3-MYB genes may be positive regulators of the HR in host in response to rust attack. This study provides a theoretical foundation for further studying these transcriptional factors in the control of the HR and the possible usage of these genes for the production of rust-resistant poplar.