Soybean (Glycine max) is a vital food crop, serving as a major source of high-quality protein for human and animal consumption. Stem diameter is one of the primary determinants of the stem lodging resistance of a given plant, but there has been relatively little research to date focused on genes associated with this trait. To address this gap in the literature, 207 chromosome segment substitution lines (CSSLs) were generated in the present study through the crossing and backcrossing of the improved Suinong14 and the wild ZYD00006 soybean varieties. These CSSLs were then used for the mapping of quantitative trait loci (QTLs) associated with stem diameter in two-year field planting materials, leading to the identification of nine QTLs. Whole genome resequencing, RNA-seq, and qPCR were then used to evaluate candidate genes associated with stem diameter within these QTL intervals, ultimately leading to the selection of Glyma.04G004100 as a stem diameter-related gene. Subsequent qPCR analyses revealed that Glyma.04g004100 was upregulated in soybean plants with larger stem diameters, and haplotype analyses yielded results consistent with these stem diameter data in the population used to conduct this study. In summary, a series of QTLs associated with stem diameter were identified in the present study, resulting in the establishment of Glyma.04g004100 as a stem diameter-related gene. Together, these results offer a theoretical foundation for the future molecular-assisted breeding of lodging-resistant soybean varieties, and future functional research focused on Glyma.04g004100 may elucidate the molecular mechanisms and key signaling networks involved in soybean stem development.