The cyanogenic glucoside dhurrin is found in sorghum and is known for its role in defense against biotic and abiotic stresses, both involving hydrogen cyanide (HCN) release. The fungus Sporisorium reilianum f. sp. reilianum (SRS) causes sorghum head smut, with infection occurring at the seedling stage and later leading to panicle loss. This video focuses on determining the role of dhurrin in sorghum’s reaction against SRS infection. It investigates the genomic basis of HCN potential (HCNp) variation and its relationship with seedlings’ response to SRS inoculation, along with other sorghum traits, and examines the expression of dhurrin biosynthetic genes in SRS-inoculated young sorghum. Genome-wide association studies (GWAS) using HCNp scores reveal significant single nucleotide polymorphisms (SNPs) on chromosomes harboring the dhurrin biosynthetic and catabolic genes, though not in close proximity. Significant hits are also detected in or near genes encoding proteins involved in plant defense against biotic stresses. Correlation analyses show a strong positive relationship between average HCNp scores and the latent period in SRS-inoculated sorghum seedlings. RT-qPCR results indicate that the dhurrin biosynthetic genes are upregulated in the leaves of the head smut resistant line BTx635 up to two days after SRS inoculation. The findings suggest the involvement of dhurrin in sorghum’s protection against SRS.