The aim of this work was to identify plant resistance genes to the sorghum anthracnose fungus Colletotrichum sublineolum. cDNA-AFLP transcript profiling on two contrasting sorghum genotypes inoculated with C. sublineolum generated about 3,000 informative fragments. In a final set of 126 sequenced genes, 15 were identified as biotic stress related. Seven of the plant-derived genes were selected for functional analysis using a Brome mosaic virus-based virus-induced gene silencing (VIGS) system followed by fungal inoculation and quantitative real-time PCR analysis. The candidate set comprised genes encoding resistance proteins (Cs1A, Cs2A), a lipid transfer protein (SbLTP1), a zinc finger-like transcription factor (SbZnTF1), a rice defensin-like homolog (SbDEFL1), a cell death related protein (SbCDL1), and an unknown gene harboring a casein kinase 2-like domain (SbCK2). Our results demonstrate that down-regulation of Cs1A, Cs2A, SbLTP1, SbZnF1 and SbCD1 via VIGS, significantly compromised the resistance response while milder effects were observed with SbDEFL1 and SbCK2. Expanded genome analysis revealed that Cs1A and Cs2A genes are located in two different loci on chromosome 9 closely linked with duplicated genes Cs1B and Cs2B, respectively. The nucleotide binding-leucine rich repeat (NB-LRR) encoding Cs gene sequence information is presently employed in regional breeding programs.