Wheat (Triticum spp. L.), rye (Secale cereal L.), and barley (Hordeum vulgare L.) seeds contain peptides toxic to celiac patients. Maize (Zea mays L.) and rice (Oryza sativa L.) are distant relatives of wheat as well as sorghum (Sorghum bicolor (L.) Moench) and are known to be safe for celiacs. Both immunochemical studies and in vitro and in vivo challenge of wheat-free sorghum food products support this conclusion, although molecular evidence is missing. The goal of the present study was to provide biochemical and genetic evidence that sorghum is safe for celiac patients. In silico analysis of the recently published sorghum genome predicts that sorghum does not contain peptides that are toxic for celiac patients. Aqueous/alcohol-soluble prolamins (kafirins) from different sorghum varieties, including pure lines and hybrids, were evaluated by SDS-PAGE and HPLC analyses as well as an established enzyme-linked immunosorbent assay (ELISA) based on the R5 antibody. These analyses provide molecular evidence for the absence of toxic gliadin-like peptides in sorghum, confirming that sorghum can be definitively considered safe for consumption by people with celiac disease.