Abstract: The pearl oyster Pinctada margaritifera holds high ecological and economic value in the tropical island-reef waters. However, the wild populations have declined sharply due to overfishing, habitat degradation, and climate change, necessitating basic biological studies on reproduction to support resource conservation and aquaculture. This study aims to elucidate the gonadal development patterns and ultrastructural characteristics of male and female gametes in a wild population of P. margaritifera from the island reef waters of the South China Sea, providing basic biological data for the resource conservation, artificial breeding, and ecological utilization of pearl oysters in the region. Wild P. margaritifera individuals were collected from Sanya Bay, Hainan Province. Histological sections stained with hematoxylin-eosin (H.E) were used to observe gonadal characteristics at different developmental stages. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to analyze the ultrastructure of sperm and oocytes. Developmental dynamics were evaluated using condition index and gamete diameter. The development of both testis and ovary in P. margaritifera can be typically divided into four stages: growing, mature, spawning, and resting. The condition index remained at a relatively high and stable level (0.101–0.119) g/cm³ across all developmental stages and between sexes, with no significant difference between males and females. Oocyte diameter showed significant variation with developmental stage, reaching a peak of (43.92 ± 2.44) μm at the mature stage. During late vitellogenesis, the oocyte nucleus enlarged to form a germinal vesicle, and the cytoplasm accumulated abundant yolk granules and lipid droplets. Rough endoplasmic reticulum exhibited both elongated and annular morphologies, and microvilli on the oocyte surface formed tight adhesions with surrounding follicle cells. Mature sperm were of the primitive flagellated type, consisting of a head, midpiece, and tail. The acrosome in longitudinal section presented a specific inverted "V" shape, with pre-nuclear fossa and post-nuclear fossa. The tail displayed the classic "9+2" microtubule arrangement. The gonadal development of P. margaritifera follows a typical follicular pattern, enabling a high condition index throughout the breeding season through dynamic gamete replenishment and batch spawning. Oocyte diameter serves as a reliable indicator of gonadal maturity. Vitellogenesis involves a dual mechanism combining endogenous organelle synthesis and exogenous nutrient transport via follicle cells. The specific sperm ultrastructure provides cytological evidence for the phylogeny of the suborder Pteriomorphia. This study enriches the basic reproductive biology data of P. margaritifera and other island-reef bivalves in the South China Sea, and has important guiding significance for wild population restoration and the establishment of ecological aquaculture models.