Soybean male sterile lines play vital value for developing soybean heterosis, but the three-line hybridization has some problems such as limited source of restorer lines, while environmental sensitive genic males sterile (EGMS) can change fertility under different conditions, which is a good solution to this problem. Based on previous reports that ms3 may be an EGMS material, the phenotypes and mutation sites of three independent mutants of ms3 (Washington), ms3 (Flanagan) and ms3 (Plainview) were further studied in this paper. The results showed that only sporadic black pollen grains with irregular shape were stained by I₂-KI dye in the anthers of these three mutants. High-through sequencing results showed that the mutated site of ms3 (Washington) and ms3 (Flanagan) were consistent with previous reports named ms3-1, with a large fragment insertion occurred in the PHD coding region of the third exon of MS3, leading to the destruction of the PHD domain of MS3 protein. ms3 (Plainview) is missing an A in the first exon of MS3, resulting in frameshift mutation with an open read frame encoding only 40 amino acids, complete loss of protein function, this allele is named ms3-2. semi-thin section analysis also showed that the tapetum layer and pollen development of ms3 (Plainview) were abnormal in the middle and late anther development. In addition, we designed molecular markers for the detection of ms3-1 and ms3-2 genotypes. In summary, the results of this study provide tools and materials for the application and transformation of ms3.