Alkaline stress is one of the major abiotic constraints limiting the plant growth and development. The alkaline tolerance in crop plants is a complex trait regulated by many genes. The genetic basis of alkaline tolerance is largely unknown in soybean. In this study, an integrated strategy of genomic analysis was utilized to elucidate the genetic architecture of alkaline tolerance across the panel of 326 diverse soybean cultivars. Our study revealed 28 SNPs was significantly associated with alkaline tolerance through association mapping using seven GWAS models. By considering the detection of SNPs in multiple environments and GWAS models, the genomic regions of five consistent SNPs were depicted as stable QTLs viz., qAT1, qAT4, qAT14, qAT18 and qAT20. All these five alkaline tolerance QTLs are reported for the first time. By using the in silico analysis, we detected 17 genes within physical intervals of five QTLs as putative candidates. Haplotype alleles of four candidate genes showed significant differences in the regulation of alkaline tolerance-related traits varying from two to four regulating alkaline tolerance from lowest to highest through moderate levels. The outcome of the current investigation can be used in soybean breeding programs for producing cultivars with enhanced alkaline tolerance.