[Objective] In this study, the difference of female and male Populus cathayana phyllosphere microbial community structure, and their key environmetal factors in driving the differences were investigated.[Methods] The natural Populus cathayana forest of Xiaowutai Mountain in Hebei province was chosen to explore the phyllosphere bacterial and fungal community composition of female and male Populus cathayana using 16S rRNA/ITS1 gene-based MiSeq sequencing, and their relationships with leaves physicochemical properties.[Results] The microbial diversity indexes showed no significant difference between female and male phyllosphere microbial communities (P>0.05). Significant differences in the relative abundance of bacterial genera Amnibacterium and Spingomonas (P<0.05), and fungal genera Aureobasidium, Elmerina, Exobasidium, Endoconidioma, Monilinia and Rhodotorula were detected between sexes by Metastats analysis. The microbiota analysis based on OTUs showed that both male and female of Populus cathayana had their unique phyllosphere bacteria and fungi microbiota, such as fungi Pringsheimia (0.15%) and bacteria Chitinophaga (0.04%) in the female phyllosphere. Redundancy analysis indicated the leaves moisture content significantly influenced phyllosphere fungal community structure of Populus cathayana (P<0.05), while no significant correlation was found between the phyllosphere bacterial community structure and the leaves physicochemical properties.[Conclusion] There were significant differences in relative abundance of phyllosphere bacterial and fungal at genus level between female and male Populus cathayana, and the structure of phyllosphere microbial community of different plants might be affected by the leaves physicochemical properties, which provided insight for exploring the differences of phyllosphere microorganism between dioecious plants.