By comparing the transcriptome data of male and female flower buds of Juglans maudsurica, we investigated the genetic regulation differences in male and female flower bud development and identified key genes. In this study, male and female flower buds of the male and female precocious type at the physiological differentiation stage were used as test materials. Through transcriptome sequencing data analysis, a total of 3,501 differentially expressed genes (DEGs) were identified, including 2,236 up-regulated genes and 2,184 down-regulated genes(Differentially expressed genes overlap between distinct comparison groups). Enrichment function and metabolic pathway analyses revealed that the sex differentiation process J. mandshurica flower buds involves complex biological processes and regulatory mechanisms. The phytohormone signal transduction pathway was notably enriched in all the control groups, indicating its crucial role in the process of sex differentiation. Furthermore, the number of up-regulated and down-regulated genes varied among the different controls, suggesting the presence of inhibitory effect genes during flower bud development. Using bidirectional clustering heat map analysis, we identified 11 key genes associated with sex differentiation in female-first flower buds and 10 key genes associated with sex differentiation in male-first flower buds. Overall, this study uncovers genetic regulatory differences between female-predominant and male-predominant flower buds during J. mandshurica flower bud development, and identifies several key genes and metabolic pathways associated with flower bud and sex differentiation. These findings contribute to a better understanding of the biological regulatory mechanisms underlying bud sex differentiation.