Abstract: Bennett's Stingray (Hemitrygon bennettii), an ovoviviparous benthic cartilaginous fish belonging to the family Dasyatidae, plays an important role in maintaining marine ecological balance and has certain medicinal value. However, its population has declined sharply due to marine environmental degradation and bycatch, and it has been listed as a vulnerable species by the IUCN. At present, systematic research on H. bennettii at the molecular genetic level is relatively lacking, which limits the in-depth analysis of its conservation genetics and adaptive mechanisms.In this study, high-throughput sequencing technology was used to obtain the whole-genome sequencing data of H. bennettii, and bioinformatics methods were employed for genome assembly, annotation and comparative genomic analysis. The results showed that the estimated genome size of H. bennettii was about 4 061.75 Mb, with a heterozygosity rate of 0.81% and a repeat sequence ratio of 65.38%. After updated assembly, the total genome length was 3 198.64 Mb, with a Scaffold N50 of 136.64 Mb and a BUSCO completeness of 97.02%, indicating high continuity and completeness. A total of 25 890 protein-coding genes and 7 401 non-coding RNA (ncRNA) genes were annotated, and the total length of repetitive sequences accounted for 57.50% of the genome, with LINE transposons as the dominant type.Comparative genomic analysis with 14 other cartilaginous fish species showed that H. bennettii was most closely related to red stingray (Hemitrygon akajei), with a divergence time of about 4.3 million years ago. A total of 25 unique gene families were identified in H. bennettii, including 5 significantly expanded and 1 875 significantly contracted gene families. The expanded gene families were mainly enriched in chromatin structure and gene expression regulation, while the contracted ones were related to immune response and signal transduction. In addition, 50 positively selected genes were detected, which were significantly enriched in lipid metabolism and chromatin regulation pathways, revealing the adaptive evolutionary characteristics of H. bennettii to benthic life.This study reports the high-quality reference genome of H. bennettii for the first time, providing key basic data and a research framework for subsequent studies on conservation genomics, population genetics and adaptive evolution of H. bennettii, and also providing an important supplement for the genomic research of Dasyatidae fishes in Myliobatiformes.