Technical Abstract: Powdery mildew (Microsphaera diffusa Cooke & Peck) is a common disease of soybean in many soybean growing regions of the world and under greenhouse conditions. The previously reported Rmd locus of soybean for resistance to powdery mildew was mapped on soybean molecular linkage group J (chromosome 16). We have discovered a single dominant gene in PI 243540 that provides season long resistance to powdery mildew. The objective of this study was to map the powdery mildew resistance gene in PI 243540 with polymerase chain reaction (PCR) based molecular markers. One hundred eighty four F2 plant and their F2:3 families from a cross between a powdery mildew susceptible cultivar Wyandot and PI 243540 were screened with M. diffusa in greenhouses. Bulked segregant analysis (BSA) with SSR markers were used to identify the tentative genomic location of the gene. The BSA technique localized the gene to a genomic region in soybean linkage group (LG) J. A linkage map with seven SSR and six SNP markers flanking the gene was constructed. We positioned the gene between Sat_224 and Barc-021875-04228 with a distance of 9.6 and 1.3 cM from the markers, respectively. The position of the gene was slightly different from previously reported map positions of the only known Rmd locus. We have mapped a single dominant gene, tentatively called Rmd-PI243540, near the previously known Rmd locus on chromosome 16. The molecular markers flanking the gene will be useful for marker assisted selection of this gene.