Abstract: Betula platyphylla is a monoecious tree species with unisexual flowers, which plays an important role in the study of floral development of woody plants. This study aims to initially identify gene expression and regulation during the critical periods of female flower development in B. platyphylla, megasporogenesis (prophase) and gametophyte development (metaphase), and to discover genes related to female floral development. Firstly, we ascertained the prophase and metaphase in female floral development by the cytological method. Secondly, the establishment of forward and reverse suppression subtractive hybridization (SSH) cDNA libraries BHHQ and BHHZ were carried out with the cDNA of prophase and metaphase female inflorescence as tester and driver respectively, using SSH technique. A total of 1406 high quality EST sequences were obtained, 787 of them having high homologies with the nucleic acid or protein sequences in databases and 47 genes possibly associating with flower development. The results of GO analysis showed that 34.96% of these genes are mainly involved in biosynthesis process, 16.45% in cellular component and 48.59% in molecular function. Six flower development-related genes were analyzed by qRT-PCR analysis and the results showed that each of these genes has some expression in different tissues. Among them, the expression level of bhhq.408,bhhq.435,bhhz.359, and bhhz.215 in reproductive organs was significantly higher than that in other organs, respectively; both bhhq.254 and bhhq.190 have some expression in most tissues, confirming that these genes are not only involved in the development of reproductive organs, but also play some roles in vegetative organs.