Powdery mildew (PMD), caused by the fungus Microsphaera diffusa Cooke & Peck, leads to considerable yield losses in soybean [Glycine max (L.) Merr.] under favourable environmental conditions and can be controlled by identifying germplasm resources with resistance genes. In this study, resistance to M. diffusa among resistant varieties B3, Fudou234, and B13 is mapped as a single Mendelian locus using three mapping populations derived from crossing susceptible with resistant cultivars. The position of the PMD resistance locus in B3 is located between simple sequence repeat (SSR) markers GMES6959 and Satt_393 on chromosome 16, at genetic distances of 7.1 cM and 4.6 cM, respectively. To more finely map the PMD resistance gene, a high-density genetic map was constructed using 248 F8 recombinant inbred lines derived from a cross of Guizao1 × B13. The final map includes 3748 bins and is 3031.9 cM in length, with an average distance of 0.81 cM between adjacent markers. This genotypic analysis resulted in the precise delineation of the B13 PMD resistance locus to a 188.06-kb genomic region on chromosome 16 that harbours 28 genes, including 17 disease resistance (R)-like genes in the reference Williams 82 genome. Quantitative real-time PCR assays of possible candidate genes revealed differences in the expression levels of 9 R-like genes between the resistant and susceptible parents. These results provide useful information for marker-assisted breeding and gene cloning for PMD resistance.