The females and males of dioecious plants have evolved sex-specific characteristics in terms of their morphological and physiological properties. However, the differentiation of phyllosphere microorganism of dioecious plants between parents and hybrid offspring remain largely unexplored. Here, the phyllosphere bacterial and fungal community diversity and composition of female (Populus nigra ‘DH5’ (PNDH5)), male (P. simonii ‘DH4’ (PSDH4)), and the hybrid offspring (P. simonii × P. nigra ‘DH1’ (PSPNDH1), P. simonii × P. nigra ‘DH2’ (PSPNDH2), P. simonii × P. nigra ‘DH3’ (PSPNDH3)) were investigated using 16S rDNA/ITS rDNA gene-based Illumina NovaSeq 6000 sequencing. There was considerable variation of plant height, diameter at breast height, leaf area, length of petioles, leaf moisture content, and starch among different samples, and PSDH2 owned the highest plant height, diameter at breast height, and length of petioles. No distinct differences of phyllosphere bacterial community diversity were observed among PSDH4, PNDH5, PSPNDH1, PSPNDH2, and PSPNDH3; while, PSPNDH2 owned the highest fungal Pielou_e index, Shannon index, and Simpson index. Firmicutes and Ascomycota were the predominant phyllosphere bacterial and fungal community at the phylum level, respectively. Bacilli and Gammaproteobacteria were the two most dominant bacterial classes regardless of parent and the hybrid offspring. The predominant phyllosphere fungal community was Dothideomycetes at the class level. The NMDS demonstrated that phyllosphere microbial community obviously differed between parents and offspring, while the phyllosphere microbial community presented some similarities under different hybrid progeny. Also, leaf characteristics contributed to the differentiation of phyllosphere bacterial and fungal communities between parents and hybrid offspring. These results highlighted the discrimination of phyllosphere microorganisms on parent and hybrid offspring, which provided clues to potential host-related species in the phyllosphere environment.