Rice grain oil is a valuable nutrient source. However, the genetic basis of oil biosynthesis in rice grains remains unclear. In this study, we performed a genome-wide association study on oil composition and oil concentration in a diverse panel of 533 cultivated rice accessions. High variation for 11 oil-related traits was observed, and the oil composition of rice grains showed differentiation among the subpopulations. We identified 46 loci that are significantly associated with grain oil concentration or composition, 16 of which were detected in three recombinant inbred line populations. Twenty-six candidate genes encoding enzymes involved in oil metabolism were identified from these 46 loci, four of which ( PAL6, LIN6, MYR2, and ARA6) were found to contribute to natural variation in oil composition and to show differentiation among the subpopulations. Interestingly, population genetic analyses revealed that specific haplotypes of PAL6 and LIN6 have been selected in japonica rice. Based on these results, we propose a possible oil biosynthetic pathway in rice grains. Collectively, our results provide new insights into the genetic basis of oil biosynthesis in rice grains and can facilitate marker-based breeding of rice varieties with enhanced oil and grain quality.