Seed-flooding stress is one of major abiotic constraints that adversely affects soybean production worldwide. Identifying tolerant germplasms and revealing the genetic basis of seed-flooding tolerance are imperative goals for soybean breeding. In the present study, high-density linkage maps of two inter-specific recombinant inbred line (RIL) populations, named NJIRNP and NJIR4P, were utilized to identify major quantitative trait loci (QTLs) for seed-flooding tolerance using three parameters viz., germination rate (GR), normal seedling rate (NSR), and electrical conductivity (EC). A total of 25 and 18 QTLs were detected by composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively, and 12 common QTLs were identified through both methods. All favorable alleles for the tolerance are notably from the wild soybean parent. Moreover, four digenic epistatic QTL pairs were identified, and three of them showed no main effects. In addition, the pigmented soybean genotypes exhibited high seed-flooding tolerance compared with yellow seed coat genotypes in both populations. Moreover, out of five identified QTLs, one major region containing multiple QTLs associated with all three traits was identified on Chromosome 8, and most of the QTLs within this hotspot were major loci (R2> 10) and detectable in both populations and multiple environments. Based on the gene expression and functional annotation information, 10 candidate genes from QTL “hotspot 8-2” were screened for further analysis. Furthermore, the results of qRT-PCR and sequence analysis revealed that only one gene,GmDREB2(Glyma.08G137600), was significantly induced under flooding stress and displayed a TTC tribasic insertion mutation of the nucleotide sequence in the tolerant wild parent (PI342618B).GmDREB2encodes an ERF transcription factor, and the subcellular localization analysis using green fluorescent protein (GFP) revealed thatGmDREB2protein was localized in the nucleus and plasma membrane. Furthermore, overexpression ofGmDREB2significantly promoted the growth of soybean hairy roots, which might indicate its critical role in seed-flooding stress. Thus,GmDREB2was considered as the most possible candidate gene for seed-flooding tolerance.