The maize male-sterile 20s2 （ms20s2） is a pollen-free genic male sterility mutant that was identified in maize inbred line KWS49. Compared with wild type （WT）， the mutant anthers were small and whitish without pollen grains. The scanning electron microscopy （SEM） observations showed that no pollen mother cells undergoing meiosis were observed in the anthers of the ms20s2 at V9 stage. The anther cuticular was abnormal， and failed to generate ubisch bodies on the inner surface of the anther wall of ms20s2 at tasseling stage. By analyzing the paraffin sections of anthers from different developmental stages， some middle layer cells and tapetum cells of the ms20s2 anther underwent abnormal division from S6 to S7 stages compared to WT， leading to the anther wall shrinking， abnormal meiosis and death of the pollen mother cells， and finally male sterility. The segregation analysis in an F₂ population revealed that the male sterility of the ms20s2 mutant was controlled by a single recessive nuclear gene. By genotyping with the maize 10K SNP chip， the causal gene was preliminarily mapped to the 6.21 Mb region on the long arm of chromosome 2. The physical interval was further delimited to 590 kb， where a known protein-coding gene MS32 （Zm00001eb106620） is present. Sequencing analysis of the MS32 gene revealed a 3166 bp insertion in the exon 4 of ms20s2， and this insertion might result in abnormal anther development and male sterility. Allelism test showed that the ms20s2 was a new allelic variation of maize male sterile gene MS32. The MS32 gene was expressed in maize anthers at S6 and S7 stages， which provided additional evidence in regulating the development of tapetum and middle layer of maize anthers.