Abstract: 【Objective】To identify candidate genes and signaling pathways associated with gonadal development in Tylorrhynchus heterochaetus， aiming to elucidate the molecular mechanisms underlying sexual maturation， which provided a theoretical foundation for advancing large-scale artificial breeding techniques for T. heterochaetus. 【Method】The study involved the examination of mature male （M）， mature female （F）， and undifferentiated T. heterochaetus （R） specimens. High-throughput transcriptome sequencing was conducted using the Illumina NovaSeq 6000 platform. Following data filtration， quality control， and splicing assembly， differentially expressed genes （DEGs） were identified using DESeq with criteria of Fold Change ≥2 and false discovery rate（FDR） <0.01. DEGs were further analyzed through GO functional annotation and KEGG pathway enrichment. The sequencing results were validated using real-time fluorescence quantitative PCR. 【Result】Transcriptome sequencing analysis yielded 46891 unigenes from 9 T. heterochaetus samples， and 27878 unigenes were annotated in 9 major functional databases such as COG， GO， KEGG， KOG， Pfam， Swiss-Prot， TrEMBL， eggNOG and Nr. According to the screening criteria， a total of 1798 DEGs were identified， including 349 DEGs in M vs F group， 1090 DEGs in R vs F group， and 359 DEGs in R vs M group. GO functional annotation indicated that DEGs were primarily associated with cell， cell parts， binding， cellular processes， and single biological processes. KEGG pathway enrichment analysis revealed that DEGs enriched in pathways such as oxidative phosphorylation， arachidonic acid metabolism， retinol metabolism， and glycine/serine/threonine metabolism. Fourteen DEGs related to gonadal development including DMRT1， SOX7， HSP90 and CALM were identified through KEGG pathway enrichment and BLASTx analysis of the Nr database. The expression trend of the target gene detected by real-time fluorescence quantitative PCR was basically consistent with the results of transcriptome sequencing analysis， which further confirmed the accuracy of transcriptome sequencing results. 【Conclusion】The mechanism underlying gonadal development and maturation involves the interplay of multiple genes and signaling pathways. This includes male-biased genes such as MT-CYB， HSP60 and DNAH5， and female-biased genes like HSP90， SOX7 and COL1A1， as well as oxidative phosphorylation， arachidonic acid metabolism， retinol metabolism， glycine / serine / threonine metabolism pathways related to gonadal development.