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The estuarine oyster (Crassostrea ariakensis), a species native to Asia, is widespread throughout the estuarine region along the northern and southern coasts of China and has important ecological and economic values. Owing to runoff dropping and environmental threats caused by climate change and anthropogenic influences, the natural resources of C. ariakensis have persistently declined. Therefore, breeding oysters with high adaptability, which requires efficient and accurate genotyping tools, is beneficial to restore the germplasm resources and aquaculture. However, targeted genotyping tools are poorly developed for this species and even in other aquatic species. Previously, we have detected some selective regions and genes associated with temperature and salinity adaptation using selection signature analysis and found that the upstream regulatory regions of the environment-responsive genes exhibited higher differentiation. In this study, we developed a single nucleotide polymorphism (SNP) targeted genotyping tool for C. ariakensis , which includes 30 K SNPs located in previously identified target regions. The target genomic regions were enriched by hybridization capture using double-stranded DNA (dsDNA) biotin-labeled probes, and sequenced with high depth. A total of 24,541 (81.8%) high-quality SNPs were successfully genotyped in 655 samples. Compared to the genotypes obtained from previous genome-wide resequencing, 74.2% of the SNPs had a consistency rate of 1, with an average consistency rate for each sample of 97.8%. Population structure analysis showed three divergent populations (northern, southern, and middle) on the coast of China, consistent with those constructed using SNPs derived from whole-genome resequencing data. Most of the 30 K SNPs were located in the intergenic and regulatory regions, whereas 7% were located in the coding region. These high-quality SNPs were located in 953 genes, including 100 heat-response and 348 high-salinity-responsive genes. Therefore, this panel can be used to evaluate the adaptive divergence between populations, and causative SNPs related to temperature and salinity adaptation in the linkage and association analysis, providing robust support for the molecular breeding of estuarine oyster. To the best of our knowledge, this study proposes the first high-throughput SNP-targeted genotyping tool for studying the genetic basis of environmental adaptation in oyster, and providing insights into the development of targeted SNP genotyping tools for the adaptation study in other aquatic species under climate changes. • We developed a 30 K targeted genotyping tool for Crassostrea. Ariakensis. • 24,541 high-quality SNPs were successfully genotyped in 655 samples. • These high-quality SNPs were located in environment-responsive genes. • 74.2% of the SNPs had a consistency rate of 1 with resequencing. Population structure analysis showed three divergent populations on the coast of China • This study could be used to dissect the genetic basis of environmental adaptation. [ABSTRACT FROM AUTHOR]