Fatty acid composition and its proportions are critical to the nutritional value and storage quality of rice (Oryza sativa L.) as the third major nutrient component in this staple food. This study involved crossing an indica rice variety, Huazhan (HZ), as the male parent, with a japonica variety, Nekken2, as the female parent, to produce the F1 generation. Subsequently, a population of 120 recombinant inbred lines (RILs) was developed through multiple generations of self-breeding. By utilizing a high-density molecular genetic linkage map and phenotypic data of four fatty acid components, we identified a total of 14 quantitative trait loci (QTLs) related to fatty acid composition across chromosomes 1, 3, 4, 6, 8, and 9. These included two QTLs for C₁₄ content, three for C_16:0 content, six for C_18:1 content, and three for C_18:2 content. Notably, the QTL qCOPT4.2 exhibited a high LOD score of 5.22. Within QTL intervals, genes such as OsACX3 and SLG affecting grain length were identified. The expression of candidate genes within these intervals was assessed and further analyzed by using quantitative real-time PCR. Genes such as LOC_Os01g15000, LOC_Os04g47120, LOC_Os04g49194, LOC_Os06g22080, LOC_Os06g23870, LOC_Os06g24704, LOC_Os06g30780, LOC_Os08g44840, and LOC_Os09g36860 were found to regulate fatty acid synthesis or metabolic pathways, potentially enhancing fatty acid content in rice. These QTLs are indispensable for breeding rice varieties with improved fatty acid profiles, offering new genetic resources for enhancing the nutritional and storage qualities of rice.