Water-soluble protein content (WSPC) is a critical factor in both soybean protein quality and functionality. However, the underlying genetic determinants are unclear. Here, we used 219 soybean accessions and 152 recombinant inbred lines genotyped with high-density markers and phenotyped in multi-environments to dissect the genetic architectures of WSPC and protein content (PC) using single- and multi-locus genome-wide association studies. In the result, a total of 32 significant loci, including 10 novel loci, significantly associated with WSPC and PC across multi-environments were identified, which were subsequently validated by linkage mapping. Among these loci, only four exhibited pleiotropic effects for PC and WSPC, explaining the low correlation coefficient between the two traits. The largest-effect WSPC-specific loci, GqWSPC8, was stably identified across all six environments and tagged to a linkage disequilibrium block comprising two promising candidate genes AAP8 and 2 S albumin, which might contribute to the high level of WSPC in some soybean varieties. In addition, two genes, Glyma.13G123500 and Glyma.13G194400 with relatively high expression levels at seed development stage compared with other tissues were regarded as promising candidates associated with the PC and WSPC, respectively. Our results provide new insights into the genetic basis of WSPC affecting soybean protein quality and yield.