Soybean male sterile lines play vital value for developing hybrid soybean varieties， while the traditional three-line hybridization system has problems due to a limited number of restorer lines. The environmental sensitive genic male sterile （EGMS） line can switch fertility under different conditions. In this research， the phenotypes and mutation sites of the three independent mutants ms3 （Washington）， ms3 （Flanagan） and ms3 （Plainview） were further studied. Phenotypic identification of sterility， high-throughput sequencing， molecular marker design and mutation site verification， analysis of the effect of mutation on MS3 protein structure， ms3 （Plainview） anther semi-thin section experiments were carried out separately. These results showed that most of the pollens are aborted， only sporadic pollen grains with irregular shape were stained by I₂-KI dye in the anthers of the three mutants. High-throughput sequencing results showed that ms3 （Washington） and ms3 （Flanagan） had a large fragment insertion in the PHD coding region of the third exon of the gene MS3， eventually compromising the PHD domain of MS3 protein， which was named ms3-1. In ms3 （Plainview）， there is one base pair deletion in the first exon of MS3， resulting in frameshift mutation encoding only 40 amino acids. The loss-of-function allele was named ms3-2. Through semi-thin section analysis of ms3 （Plainview）， its tapetum layer and pollen development were abnormal at the middle and late anther development stage. In summary， this study provided materials and basis for future application and genetic modification of ms3 and MS3 in soybean breeding.