As climate change impacts marine ecosystems, the production of ω-3 polyunsaturated fatty acids (PUFAs) is significantly declining. To enhance ω-3 PUFA production through genetic approaches, understanding the regulatory mechanisms is essential. Oysters, as representative bivalves, exhibit high levels of PUFAs. Two closely related species, Crassostrea gigas (adapted to cold climates) and Crassostrea angulata (adapted to warm climates), provide an opportunity to investigate the genetic and environmental effects of climate change on oysters and explore the regulatory mechanisms underlying ω-3 fatty acid dynamics. To study this, we utilized an oyster liquid 100KSNP microarray to identify fatty acid content, including C18:3ω3, EPA, and DHA. A genome-wide association study was conducted using an F2 hybridization population of 458 individuals. We mapped 344 SNPs and identified 742 candidate genes. Further analysis enriched 24 key genes from these candidates using KEGG analysis. To validate our findings, we performed GS-assisted GWAS, dominant genotype analysis, and resequencing of wild populations of C. gigas and C. angulata, confirming 23 out of the 24 key genes. Additionally, we identified 458 differentially expressed genes, including 266 between high and low EPA+DHA groups and 192 between wild C. gigas and C. angulata groups. Among the 742 associated genes and the 292 previously identified in selective sweeps, 152 showed differential expression. We constructed a regulatory network encompassing the cAMP/WNT/PI3K-AKT/AMPK signaling pathway, revealing the complex dynamics of oyster ω-3 fatty acid content. This study provides insights into the sustainable utilization of ω-3 fatty acid resources and strategies to mitigate climate change impacts.