Powdery mildew (PM), caused by the fungus Microsphaera diffusa, causes severe yield losses in soybean [Glycine max (L.) Merr.] under suitable environmental conditions. Identifying resistance genes and developing resistant cultivars may prevent soybean PM damage. In this study, analysis of F1, F2, and F8:11 recombinant inbred line (RIL) populations derived from the cross between Zhonghuang 24 (ZH24) and Huaxia 3 (HX3) indicated that adult-plant resistance (APR) to powdery mildew in the soybean cultivar (cv.) ZH24 was controlled by a single dominant locus. A high-density genetic linkage map of the RIL population was used for fine mapping. The APR locus in ZH24 was mapped to a 281-kb genomic region on chromosome 16. Using 283 susceptible plants of another F2 population, the candidate region was fine-mapped to a 32.8-kb genomic interval flanked by the markers InDel14 and Gm16_428. The interval harbored five genes, including four disease resistance (R)-like genes, according to the Williams 82.a2.v1 reference genome. Quantitative real-time PCR assays of candidate genes revealed that the expression levels of Glyma.16g214300 and Glyma.16g214500 were changed by M. diffusa infection and might be involved in disease defense. Rmd_B13 showed all-stage resistance (ASR) to PM in soybean cv. B13. An allelism test in the F2 segregating population from the cross of ZH24 × B13 suggested that the APR locus Rmd_ZH24 and the ASR locus Rmd_B13 may be allelic or tightly linked. These results provide a reference marker-assisted selection in breeding programs.