AbstractThis study aimed to investigate the genetics of sucrose and soluble sugar accumulation in soybean [Glycine max (L.) Merrill.] and the impact on food quality. A recombinant inbred line population, constructed from ZD27 and HF25 with parents having significantly different levels of sucrose and soluble sugar content, was used. Using the QTL‐Sequencing method, we identified 16 QTLs affecting sucrose and soluble sugar, including a major sucrose‐associated QTL, qSU1901, with 10.6%–13.2% phenotypic variation for sucrose. To narrow down the list of candidate genes, we analyzed the polymorphisms within the major QTL interval. We further explored the temporal expression patterns of key genes involved in sucrose and soluble sugar transport and metabolism through dynamic transcriptome analysis. We generated a gene regulatory network that correlated with sucrose and soluble sugar content using weighted gene co‐expression network analysis. By combining QTL‐Seq and RNA‐Sequencing data, we narrowed down 233 candidate genes. In conclusion, our findings provide a better understanding of sucrose and soluble sugar regulation in soybean.