Zeaxanthin, a natural fat-soluble pigment, not only increases plant resistance, but also has vital significance for human health. However, quantitative trait loci (QTL) and the epistatic effects of zeaxanthin concentration in maize kernel have not been well studied. To identify QTLs and analyse the epistatic effects of zeaxanthin concentration in maize kernel, two sets of segregating generations derived from the cross between HuangC (a high zeaxanthin concentration inbred line) and Rezi1 (a low zeaxanthin concentration inbred line) were evaluated in three different environments. One major-effect QTL, qZea6a, explains 41.4–71.4% of the phenotypic variation and two QTLs, qZea4a and qZea3a, show LOD > 3 for zeaxanthin concentration detected over two generations and three different environments. Four of the ten QTL pairs show epistatic effects, explaining 7.34–14.3% of the phenotypic variance. Furthermore, additivity was the major allelic action at zeaxanthin concentration QTLs located in F2 and F2:3 populations and plants with homozygous HuangC alleles have a strong genetic ability in enhancing zeaxanthin concentration in maize kernel. These results will contribute to understanding these complex loci better and provide awareness about zeaxanthin concentration to maize breeders and scientists involved in maize research.