To investigate the status of germplasm resources and the level of genetic diversity of American shad (Alosa sapidissima) breeding populations in southern Jiangsu, a total of 210 individuals from seven different populations including Zhenjiang Dantu (Dtq), Suzhou Zhangjiagang (Zjg), Suzhou Xiangcheng (Xcq), Nantong Zhongyang (Zyq), Zhenjiang Yangzhong (Yzq), Changzhou Gehu (Ghq) and Changzhou Wujin (Czq) were analyzed using 15 microsatellite markers and mitochondrial D-loop sequences in this study. With the exception of Asa-12, the findings revealed that all 15 SSR loci exhibited high polymorphism (PIC > 0.5). Among them, the expected heterozygosity (H_e) of the seven populations ranged from 0.615 to 0.758, and the polymorphic information content PIC ranged from 0.568 to 0.723, both of which were highest in the Zjg population. A total of 32 mutation loci and 20 haplotypes were defined for the D-loop sequence analysis, with the Zjg population having the most haplotypes (11). The haplotype diversity and nucleotide diversity indices for the seven populations ranged from 0.618 to 0.945 and from 0.003 to 0.008, respectively. The genetic distance of Nei's (0.058) and the genetic K2P distance (0.003) between the Ghq and Zyq populations, determined by genetic distance analysis using SSR and D-loop sequences, were the lowest and were lower than the genetic distances between other populations (0.073-0.397 and 0.003-0.006, respectively). We conducted a thorough investigation and discovered that genetic variation among the seven populations was low, despite the fact that they were genetically diverse. The gene flow N_m > 1 and the mosaic arrangement of the different evolutionary trees supported the strong connectivity of the seven groups. This study provides a theoretical basis for further breeding efforts and a preliminary understanding of the status of American shad germplasm in southern Jiangsu Province.