Maize kernel oil is a valuable source of nutrition. Here we extensively examine the genetic architecture of maize oil biosynthesis in a genome-wide association study using 1.03 million SNPs characterized in 368 maize inbred lines, including 'high-oil' lines. We identified 74 loci significantly associated with kernel oil concentration and fatty acid composition (P < 1.8 × 10(-6)), which we subsequently examined using expression quantitative trait loci (QTL) mapping, linkage mapping and coexpression analysis. More than half of the identified loci localized in mapped QTL intervals, and one-third of the candidate genes were annotated as enzymes in the oil metabolic pathway. The 26 loci associated with oil concentration could explain up to 83% of the phenotypic variation using a simple additive model. Our results provide insights into the genetic basis of oil biosynthesis in maize kernels and may facilitate marker-based breeding for oil quantity and quality.