RAD-seq technology was utilized to develop 10 pairs of novel polymorphic microsatellite primers for assessing genetic diversity in nine wild populations of Sinonovacula constricta. In 270 individuals of S. constricta, a total of 352 alleles were detected. The average number of alleles (Na) ranged from 3.2000 to 4.3000, with the average effective number of alleles (Ne) falling between 1.8789 and 2.5433. Observed heterozygosity (Ho) varied from 0.0000 to 0.9667, with an average ranging from 0.3088 to 0.5533. Expected heterozygosity (He) spanned from 0.0000 to 0.7945, with an average of 0.3456 to 0.5715. The average polymorphic information content (PIC) ranged from 0.3373 to 0.5989. Genetic diversity analysis indicated a moderate level of genetic diversity across the nine populations of S. constricta. The genetic differentiation coefficient (Fst) among populations ranged from 0.0547 to 0.3511, with the lowest Fst value observed between Qinhuangdao in Hebei and Zhuanghe in Dalian (0.0547) and the highest between Lianyungang in Jiangsu and Beihai in Guangxi (0.3511). Gene flow (Nm) values ranged from 0.4620 to 4.3204, with the highest Nm value recorded between Qinhuangdao and Zhuanghe (4.3204) and the lowest between Lianyungang and Beihai (0.4620). AMOVA analysis revealed that 33.04% of the total genetic variation existed among populations (P<0.01), while 66.96% was within populations (P<0.01), underscoring significant genetic variation both within and among populations, with greater diversity observed among individuals. Consistent results were obtained from UPGMA clustering, Structure clustering, and Principal Coordinate Analysis (PCoA). The study delineated three main groups among the nine populations: the first group comprised Qinhuangdao in Hebei and Zhuanghe in Dalian, which were closely related and later merged with Shazikou in Qingdao and Dandong in Liaoning; the second group included Lianyungang in Jiangsu, Xiangshan in Ningbo, Xiamen in Fujian, and Huizhou in Guangdong; and the third group was independently represented by Beihai in Guangxi. These findings enhance the genetic background knowledge of S. constricta, offering crucial foundational data for enhancing germplasm resource management and preventing population degradation in this species.