Smut is a serious disease in Job’s tears, also known as adlay, and contributes to the reduction of crop yield in agricultural fields.  In this study, the key pathogenic fungi in adlay smut disease were first identified by internal transcribed spacer region (ITS) high-throughput sequencing and then used to elucidate the composition and diversity of the fungal community in adlay smut.  Results indicate that an abundance of operational taxonomic units (OTUs) were detected in the infected involucres of flowering plants and the OTUs were classified to nine phyla, 20 classes, 45 orders, 90 families and 119 genera.  A total of 4 986 OTUs clustered together, sharing six core OTUs in all samples, while 145 OTUs were shared among four geographical groups.  The Shannon and Simpson indices ranged from 0.137–1.629 and 0.357–0.970, respectively.  Community diversity ranked as Anshun (AS)>Qinglong (QL)>Xingren (XR)>Xingyi (XY) among the four geographical groups by Shannon and Simpson indices, exhibiting complex community diversities among accessions and geographical groups.  The richness and diversity data imply a weak relationship between the accession community richness and geographical origins of samples.  Two closely related fungal genera, Sporisorium and Ustilago, were implicated as causes of smut disease.  The genus Sporisorium appears to be more commonly found among accessions and thus is more likely to be the fungal pathogen causing smut in adlay.  This work can facilitate strategies to control and prevent smut infection to improve adlay yield.