BACKGROUND: Crop biofortification is a successful strategy to ameliorate Vitamin A deficiency. Sorghum is a good candidate for vitamin A biofortification, as it is a staple food in regions with high prevalence of vitamin A deficiency. β-carotene-the main provitamin A carotenoid-is below the target concentration in sorghum grain, therefore biofortification breeding is required. Previous studies found evidence that sorghum carotenoid variation is oligogenic, suggesting that marker-assisted selection can be an appropriate biofortification method. However, we hypothesize that sorghum carotenoids have both oligogenic and polygenic components of variation. Genomics-assisted breeding could accelerate breeding efforts, but there exists knowledge gaps in the genetics underlying carotenoid variation, as well as appropriate germplasm to serve as donors.RESULTS: In this study, we characterized carotenoids in 446 accessions from the sorghum association panel and carotenoid panel using high-performance liquid chromatography, finding high carotenoid accessions not previously identified. Genome-wide association studies conducted with 345 accessions, confirmed that zeaxanthin epoxidase is a major gene underlying variation for not only zeaxanthin, but also lutein and β-carotene. High carotenoid lines were found to have limited genetic diversity, and originated predominantly from only one country. Potential novel genetic diversity for carotenoid content was identified through genomic predictions in 2,495 accessions of unexplored germplasm. Oligogenic variation of carotenoids was confirmed, as well as evidence for polygenic variation, suggesting both marker-assisted selection and genomic selection can facilitate breeding efforts.CONCLUSIONS: Sorghum vitamin A biofortification could be beneficial for millions of people who rely on it as a dietary staple. Carotenoid content in sorghum is low, but high heritability suggests that increasing concentrations through breeding is possible. Low genetic diversity among high carotenoid lines might be the main limitation for breeding efforts, therefore further germplasm characterization is needed to assess the feasibility of biofortification breeding. Based on germplasm here evaluated, most countries' germplasm lacks high carotenoid alleles, thus pre-breeding will be needed. A SNP marker within the zeaxanthin epoxidase gene was identified as a good candidate for use in marker-assisted selection. Due to the oligogenic and polygenic variation of sorghum grain carotenoids, both marker-assisted selection and genomic selection can be employed to accelerate breeding efforts.