<p>The orange-spotted grouper (<i>Epinephelus coioides</i>) is an economically important marine species in the South China Sea. Due to overfishing and environmental pollution, its natural resources in the South China Sea have severely declined. Evaluation of genetic diversity of the orange-spotted grouper in the distribution areas is important for future conservation action. In this study, a molecular marker specific for orange-spotted grouper <i>E. coioides</i> was developed based on the mitochondrial D-loop region sequences. We evaluated the community structure and genetic diversity of the groupers at four stock enhancement sites of the Wanshan Archipelago by employing integrated assessment methods including environmental DNA (eDNA) and the D-loop marker. Five grouper species were identified from the eDNA samples using 12S rDNA metabarcoding technology, with the orange-spotted grouper being the most abundant grouper ranging from 39.05% to 49.79% of the grouper contents. Furthermore, 15 D-loop haplotypes for the orange-spotted grouper in the Wanshan Archipelago release areas were detected by utilizing the novel developed D-loop marker. High haplotype genetic diversity was observed at all sampling sites for the orange-spotted grouper population. Dominant haplotypes such as ASV_1, ASV_2, ASV_3, ASV_4, and ASV_5 exhibited high levels of geographic sharing, suggesting a degree of ecological or environmental similarity across these regions. Most of the genetic variations were originated within populations, indicating significant genetic differentiation among grouper populations in the Wanshan Archipelago. Our study indicates that eDNA technology is a valuable non-invasive tool for monitoring fish community structure, and the haplotype diversity of <i>E. coioides</i> in the sampled waters maintained a relatively high haplotype richness.</p>