ABSTRACT: Background: The Japanese eel (Anguilla japonica) holds significant economic value in East Asia, but limitations in understanding its reproductive biology have hindered advancements in artificial breeding techniques. Previous research has primarily focused on conserved sex differentiation genes, offering limited insights into the broader molecular mechanisms driving gonadal development and sexual dimorphism. To address these limitations, this study aims to investigate key genes and pathways involved in gonadal development through a comprehensive transcriptomic analysis of male and female eel gonads. Results: PacBio Iso-Seq and Illumina RNA-Seq technologies were combined to conduct a full-length transcriptome analysis of male and female Japanese eel gonads at a post-differentiation, pre-maturation stage. A total of 24661 unigenes were identified in ovaries and 15023 in testes, along with genomic regulatory elements such as transcription factors, simple sequence repeats, and long non-coding RNAs. Additionally, 1,210 differentially expressed genes were detected. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed significant pathways involved in cell cycle regulation, metabolic processes, apoptosis, and hormone activity. Notably, several reproductive-related genes, including bambi, ccnb1, cdc20, gdf9, prlh, ccdc39, chrebp, tspo, syce3, and ngb, demonstrated significant dimorphic expression in eel gonads. Conclusions: This study provides valuable insights into the molecular mechanisms of gonadal differentiation and sexual dimorphism in Japanese eels. The findings expand the genetic resources available for the eel breeding industry and could facilitate the development of improved artificial breeding techniques focused on reproductive development.