Abstract: Soybean is an important source of vegetable protein, and innovation of high-protein soybean germplasm becomes of importance in breeding for elite soybean varieties and exploring the underlying mechanism. In order to obtain the high-protein soybean germplasm resources, the protein content of EMS mutagenized "Zp661" M2 population were measured following the NIR (Near-Infrared Reflectance) method, and the subsets of selected offspring lines were further investigated. The results showed that the protein content of 1971 M2 seeds ranged from 34.96% to 50.16% with an average of 42.97%, being 5.11% higher than wild type (40.88%). The protein content in 17.65% of tested M2 samples was higher than 45%. The mutants with high protein content showed a constant reduction in offspring lines in each of generations, whereas an increasing protein content was also observed in offspring lines of M2 mutants with low-protein content. Notably, we identified nine high protein M7 homozygous mutants (m1 ~ m9) with a mean of 48.17%, which were 16.94% higher than wild type (41.19%), with an increased 7.23% genetic gain. The mutants m1 to m7 were derived from sergeants of the M2 high-protein content mutant 20722 (47.21%), while m8 and m9 were derived from the M2 low protein content mutant 3442 (38.51%). Furthermore, that protein content in m2, m4 and m5 was found to be significantly positively correlated with 100-seed weight and single plant seed yield. Thus, the findings implied no negative correlation between protein content and yield production in some of mutants, and these germplasm resources might become valuable in future breeding for soybean varieties with simultaneous high-yield and high-quality.