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Coffee Berry Disease (CBD) caused by the fungal pathogen Colletotrichum kahawae destroys about 80% of the crop on susceptible Coffea arabica cultivars if no control is applied. Resistance to CBD is governed by three genes: T, R that are dominant, and the recessive k in varieties Hibrido de Timor, Rume Sudan (RS), and K7 respectively. The marker for the T-gene had earlier been identified. The study aimed to identify the DNA marker for the R-gene from RS. The mapping population comprised of 106 F2 genotypes from varieties RS (resistant) and SL28 (susceptible). The genotypes were assessed for their reaction to CBD using hypocotyl inoculation test on a scale of 1–12. The Single Nucleotide Polymorphism (SNP) markers were obtained through Genotyping by Sequencing. The population structure was assessed by both principal component analysis and marker-based kinship while pairwise linkage disequilibrium was estimated using squared allele frequency correlations (r2). Genome-Wide Association Study was carried out using a compressed mixed linear model in Genomic Association and Prediction Integrated Tool. Two SNP markers were significantly associated with CBD resistance in coffee Chromosomes (Chr) 1 and 2 accounting for 12.5% and 11% respectively of the total phenotypic variation explained by the model. The sequence search for the markers was associated with proteins for disease resistance within the NCBI database. The two SNP markers 100025973|F|0-59:T > C-59:T > C and 100034991|F|0-44:C > T-44:C > T are identified as DNA markers for the genetic loci conferring resistance to CBD in RS, christened as Ck-2 and Ck-3 and are recommended for marker assisted selection.