Trachycarpus fortunei (Hook.) is a typical dioecious plant, which has important economic value. There is currently no sex identification method for the early stages of T. fortunei growth. The aim of this study was to obtain expression and site differences between male and female T. fortunei transcriptomes. Using the Illumina sequencing platform, the transcriptomes of T. fortunei male and female plants were sequenced. By analyzing transcriptomic differences, the chromosomal helical binding protein (CHD1), serine/threonine protein kinase (STPK), cytochrome P450 716B1, and UPF0136 were found to be specifically expressed in T. fortunei males. After single nucleotide polymorphism (SNP) detection, a total of 12 male specific sites were found and the THUMP domain protein homologs were found to be male-biased expressed. Cytokinin dehydrogenase 6 (CKX6) was upregulated in male flowers and the lower concentrations of cytokinin (CTK) may be more conducive to male flower development. During new leaf growth, flavonoid and flavonol biosynthesis were initiated. Additionally, the flavonoids, 3',5'-hydroxylase (F3'5'H), flavonoids 3'-hydroxylase, were upregulated, which may cause the pale yellow phenotype. Based on these data, it can be concluded that inter-sex differentially expressed genes (DEGs) and specific SNP loci may be associated with sex determination in T. fortunei.