The maize male-sterile 20s2 (ms20s2) is a pollen-free genic male sterility mutant that we identified in maize inbred line KWS49. Compared with wild type (WT), the mutant anthers were small and whitish, lacking pollen grains. However, there were no significant differences in agronomic traits such as plant height and ear height between the WT and ms20s2 mutant. The scanning electron microscopy (SEM) observations showed that, compared to WT, no pollen mother cells undergoing meiosis were observed in the anthers of the ms20s2 at V9 stage; and the anther cuticular was abnormal, and no ubisch bodies were observed on the inner surface of the anther wall of ms20s2 at tasseling stage. Analysis of paraffin sections of anthers from different developmental stages showed that some middle layer cells and tapetum cells of the ms20s2 anther underwent abnormal division from S6 to S7 stages compared to WT, causing the anther wall shrinking, pollen mother cells cannot perform meiosis normally, and leading to the death of the pollen mother cells and male sterile phenotype. The genetic analysis revealed that the male sterile phenotype of the ms20s2 mutant was controlled by a single recessive nuclear gene. By analyzing the genotypes of 60 sterile plants in the F2 mapping population with the maize 10K SNP chip, the mutation was preliminarily mapped to the 6.21Mb region on the long arm of chromosome 2 of maize. After developing new InDel markers and expanding the mapping population, the gene was fine mapped to the 590kb region, which contains a known protein-coding gene MS32 (Zm00001eb106620). Sequencing analysis of MS32 gene revealed that there is a 3166bp insertion in the exon 4 of ms20s2, which may affect the protein function of MS32, resulting in abnormal anther development and male sterility phenotype of ms20s2 mutant. Allelism test showed that the ms20s2 was a new allelic variation of maize male sterile gene MS32. The tissue specific expression analysis showed that the gene was expressed in maize anthers at S6 and S7 stages, which further verified that the gene played an important role in regulating the development of tapetum and middle layer of maize anthers.