Grain mold, caused by a consortium of pathogenic fungal species, is the most important disease of sorghum [Sorghum bicolor (L.) Moench]. Genome wide association study on 1425 diverse Ethiopian sorghum landraces identified a major grain mold resistance locus containing tightly linked and sequence related MYB transcription factor genes. The locus contains YELLOW SEED1 (Y1), a likely non-functional pseudo gene (Y2), and YELLOW SEED3 (Y3). SNPs and other sequence polymorphisms that alter the Y1 and Y3 genes correlated with susceptibility to grain mold and provided a strong genetic evidence. Accordingly, the expression of both Y1 and Y3 genes in the developing grain and glumes of a widely known susceptible sorghum line, RTx430, were severely reduced but significantly increased in the resistant line, RTx2911. In addition, the expression of flavonoid biosynthesis genes such as DIHYDROFLAVONOL 4-REDUCTASE 3 (DFR3) was significantly induced in the resistant line in response to inoculation by a mixture of spores from different molding fungi while the susceptible line displayed reduced expression. The data suggest that the MYB genes and their grain and glume specific expressions may determine the differential regulation of the flavonoid biosynthesis pathway genes, the synthesis of 3-Deoxyanthocynidins and ultimately responses to molding fungi. The study also suggested that resistance to grain mold may be negatively associated with grain functionality traits such as ‘injera’ making quality of sorghum.