Soybean (Glycine max L. Merr.) is rich in proteins, fats, and other nutrients, and the genetic improvement of soybean protein content has long been a key research focus in breeding programs. Based on the chromosome segment substitution line (CSSL) population, this study screened target lines within this population using genotypic and phenotypic information to establish an initial mapping population for soybean seed protein content. Through single-marker analysis, a quantitative trait locus (QTL) interval was mapped to the region between 26,705,080 bp and 33,180,908 bp on chromosome 16, designated as qSPJ_1. A secondary segregating population was constructed based on the initial mapping results for fine mapping, which narrowed the interval to 0.076 Mb. A total of 9 candidate genes were identified within this interval. By comparing amino acid and promoter sequences between the two parents, performing quantitative real-time PCR (qRT-PCR) analysis, and conducting haplotype analysis, Glyma.16G165100 was preliminarily predicted as a candidate gene affecting soybean seed protein content. The single nucleotide polymorphism (SNP) variation sites in its promoter region were significantly associated with the variation in protein content in the resource population. This study provides important theoretical guidance for dissecting the genetic mechanism of soybean seed protein content and advancing its breeding improvement.