In plants with C4 photosynthesis, physiological mechanisms underlying variation in stable carbon isotope discrimination (Δ(13)C) are largely unknown, and genetic components influencing Δ(13)C have not been described. We analyzed a maize (Zea mays) introgression library derived from two elite parents to investigate whether Δ(13)C is under genetic control in this C4 species. High-density genotyping with the Illumina MaizeSNP50 Bead Chip was used for a detailed structural characterization of 89 introgression lines. Phenotypic analyses were conducted in the field and in the greenhouse for kernel Δ(13)C as well as plant developmental and photosynthesis-related traits. Highly heritable significant genetic variation for Δ(13)C was detected under field and greenhouse conditions. For several introgression library lines, Δ(13)C values consistently differed from the recurrent parent within and across the two phenotyping platforms. Δ(13)C was significantly associated with 22 out of 164 analyzed genomic regions, indicating a complex genetic architecture of Δ(13)C. The five genomic regions with the largest effects were located on chromosomes 1, 2, 6, 7, and 9 and explained 55% of the phenotypic variation for Δ(13)C. Plant development stage had no effect on Δ(13)C expression, as phenotypic as well as genotypic correlations between Δ(13)C, flowering time, and plant height were not significant. To our knowledge, this is the first study demonstrating Δ(13)C to be under polygenic control in the C4 species maize.