Rice sheath blight (RSB), caused by the soil-borne necrotrophic fungus Rhizoctonia solani, is a major disease that can lead to significant yield losses under favorable conditions. Resistance to RSB is a complex quantitative trait controlled by multiple genes, but no single dominant resistance gene or locus has been identified to date. This study used a Genome-Wide Association Study (GWAS) to identify genetic loci associated with RSB resistance in a panel of 200 diverse rice varieties, including traditional upland and lowland varieties originating from Malaysia. We employed a double-digest RAD sequencing (ddRAD) approach to genotype the population. Bioinformatic analysis of sequencing data from 197 samples revealed two major genetic clusters and an optimal population structure (K=3). Phenotypic screening for sheath blight resistance showed that both lesion length (PL) and relative lesion height (PJ) data were normally distributed, making them suitable for parametric statistical analysis. The GWAS successfully identified a significant locus on Chromosome 5 associated with RSB resistance. A total of four significant SNPs were annotated to the gene LOC_Os05g47770, which encodes a Serine/Threonine-Protein Kinase (STPK). This gene family is widely recognized for its role in plant defense responses. This discovery provides crucial genetic insights for developing DNA markers for marker-assisted selection (MAS) in rice breeding programs, ultimately contributing to the creation of more disease-resistant rice varieties.