Abstract: This study aimed to characterize the current distribution of spawning grounds of Coilia nasus in Poyang Lake and provide a scientific basis for the conservation of its critical habitats. Field surveys were conducted to investigate the spatial and temporal distributions of adult C. nasus, eggs, and larvae. Sex identification and gonadal developmental staging were performed. In addition, molecular markers were developed to distinguish migratory C. nasus from Coilia brachygnathus. Habitat suitability modeling was combined with field observations to identify potential spawning areas and characterize their environmental features. A total of 7 324 C. nasus specimens were collected from the main lake and the upstream sections of the Gan, Xiu, Xin, and Rao rivers between 2019 and 2021. Based on their spatial and temporal distributions and catch per unit effort (CPUE), eight major distribution areas were identified: Gutang, Huoyanshan, Huangjinzu, Piaotou, Zengmizhou, Lutan, Ruihong, and Longkou. The distribution of migratory C. nasus was significantly associated with water depth, and the highest CPUE was recorded at depths of 16-17 m. In six areas, namely Gutang, Huoyanshan, Huangjinzu, Zengmizhou, Lutan, and Ruihong, more than 89.8% of spawning adults had gonads beyond developmental Stage IV, and 30.6% had reached Stage V. These six areas were therefore identified as potential spawning areas. Eggs and larvae collected from Gutang and Huoyanshan were identified through sequencing and comparison of the RH2A opsin gene. The analysis established, for the first time, molecular markers capable of distinguishing migratory C. nasus from C. brachygnathus and confirmed Gutang and Huoyanshan as spawning grounds of C. nasus. Preliminary surveys of the topography, elevation, and flow velocity of these spawning grounds were also conducted. Poyang Lake continues to provide functional spawning habitat for C. nasus and plays an important role in the replenishment of its populations. These findings provide an important scientific basis for identifying and conserving the spawning grounds and associated habitats of C. nasus in Poyang Lake.