Three environmentally stable major QTL controlling seed oil content in peanut were identified, and their genetic effects were evaluated in near‑isogenic lines and a peanut germplasm panel. Increasing seed oil content (SOC) is a primary objective in peanut (Arachis hypogaea L.) breeding, meeting the rising global demand for edible oil. Quantitative trait loci (QTL) mapping can help to identify genes underlying SOC variation and develop markers to enhance selection efficiency by marker-assisted breeding. In the present study, three major and stable QTL for SOC were identified on peanut chromosome Arahy.08, using a bulked segregant analysis (BSA) approach based on whole-genome sequencing of F recombinant inbred lines (RILs). The QTL qSOCA08-1, explaining 11.41-20.97% of phenotypic variation, was mapped on a 0.65-Mb genomic region. The QTL qSOCA08-2, accounting 25.57-39.40% of phenotypic variation, was located on a 1.04-Mb physical interval. Finally, qSOCA08-3 explaining up to 17.31% of the phenotypic variation was mapped in a genomic region of 1.02-Mb. The genetic effects of these three QTL were assessed using near‑isogenic lines (NILs), derived from residual heterozygous individuals, and a tetraploid peanut germplasm panel. Potential candidate genes within the physical intervals of corresponding major QTL were predicted to participate in the oil biosynthesis in peanut. In summary, our study provides valuable genetic resources and tightly linked molecular markers for peanut molecular breeding aimed at improving SOC.