Maize (Zea mays L.) is a crucial global crop that serves as a primary source of food and feed. However, its kernels are susceptible to infection by Aspergillus flavus, a fungus known for producing aflatoxins-which are highly carcinogenic compounds harmful to human and animal health. Identifying quantitative trait loci (QTLs) for aflatoxin resistance and developing aflatoxin-resistant maize varieties are essential for mitigating aflatoxin contamination. In this study, a genome-wide association study (GWAS) using an enlarged genotypic panel of 311 maize inbred lines was used to identify genetic loci associated with A. flavus resistance. Phenotypic data on A. flavus resistance were collected through controlled inoculation experiments conducted under controlled conditions. The results revealed that the resistance to A. flavus follows a normal distribution. In addition, temperate inbreds exhibited stronger resistance to A. flavus than tropical/subtropical materials. This study identified 13 novel QTLs encompassing 47 highly expressed genes, with each QTL explaining 8.22–27.71% of the phenotypic variation, indicating that the higher marker density improved statistical power. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that these genes are related to fatty acid synthesis, glycoside decomposition, and root growth and development. One specific gene located on ZmAFR16, ZmFUC1, displayed clustered peaks and accounted for an average of 10.21% of the phenotypic variation. This gene was found to play a role in cell membrane formation and possess alpha-L-fucosidase activity, so it promotes glycoside metabolism and contributes to polysaccharide degradation. Haplotype analysis showed significant differences in resistance to A. flavus among the different haplotypes of elo1 and ZmFUC1. Inbreds carrying the favorable haplotype combination of these two genes exhibited strong resistance to A. flavus. A select sweep analysis indicated that ZmFUC1 was selected during the domestication of teosinte (Zea mays ssp. mexicana) to modern maize, as well as during the adaptation from tropical/subtropical maize to temperate maize. Importantly, this study developed molecular markers in the promoter region of ZmFUC1 to efficiently identify maize germplasm with beneficial haplotypes for resistance to A. flavus. These findings not only enhance our understanding of the genetic factors influencing maize kernel resistance to A. flavus but also offer valuable insights for improving existing germplasm and developing new maize varieties with enhanced resistance to this pathogen.