BACKGROUND: Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop for dryland areas in the United States and for small-holder farmers in Africa. Natural variation of sorghum grain composition (protein, fat, and starch) between accessions can be used for crop improvement, but the genetic controls are still unresolved. The goals of this study were to quantify natural variation of sorghum grain composition and to identify single-nucleotide polymorphisms (SNPs) associated with variation in grain composition concentrations.RESULTS: In this study, we quantified protein, fat, and starch in a global sorghum diversity panel using near-infrared spectroscopy (NIRS). Protein content ranged from 8.1 to 18.8%, fat content ranged from 1.0 to 4.3%, and starch content ranged from 61.7 to 71.1%. Durra and bicolor-durra sorghum from Ethiopia and India had the highest protein and fat and the lowest starch content, while kafir sorghum from USA, India, and South Africa had the lowest protein and the highest starch content. Genome-wide association studies (GWAS) identified quantitative trait loci (QTL) for sorghum protein, fat, and starch. Previously published RNAseq data was used to identify candidate genes within a GWAS QTL region. A putative alpha-amylase 3 gene, which has previously been shown to be associated with grain composition traits, was identified as a strong candidate for protein and fat variation.CONCLUSIONS: We identified promising sources of genetic material for manipulation of grain composition traits, and several loci and candidate genes that may control sorghum grain composition. This survey of grain composition in sorghum germplasm and identification of protein, fat, and starch QTL contributes to our understanding of the genetic basis of natural variation in sorghum grain nutritional traits.