A high-quality reference genome is indispensable for resolving biologically essential traits. Ficus hispida is a dioecious plant. A complete Ficus reference genome will be crucial for understanding their sex evolution and important biological characteristics, such as aerial roots, mutualistic symbiosis with ficus-wasps, and fruiting from old stems. Here, we generated a telomere-to-telomere (T2T) genome for F. hispida using PacBio HiFi and Oxford Nanopore Ultra-long sequencing technologies. The genome contiguity and completeness has shown improvement compared with the previously released genome, with the annotation of six centromeres and 28 telomeres. We have refined our previously reported 2-Mb male-specific region into a 7.2-Mb genomic region containing 51 newly predicted genes and candidate sex-determination genes AG2 and AG3. Many of these genes showed extremely low expression, likely attributed to hypermethylation in the gene body and promoter regions. Gene regulatory networks (GRNs) revealed that AG2 and AG3 are related to the regulation of stamen development in male flowers, while the AG1 gene is responsible for regulating female flowers’ defense responses and secondary metabolite processes. Comparative analysis of GRNs showed that the NAC, WRKY, and MYB transcription factor families dominate the female GRN, whereas the MADS and MYB transcription factor families are prevalent in the male GRN.

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