The diversity of fungi in wheat with different deoxynivalenol (DON) content at various periods post-harvest and in the environment of storage were investigated. The changes in DON content were measured with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS), and an amplicon sequence analysis of fungi was performed in traditional storage structures using high-throughput sequencing. The changes in temperature, humidity, and CO2 concentration were collected by sensors. In addition, we analyzed principal component analysis, species composition, species differences, and community differences of fungi. There was an obvious separation of the fungal communities under different storage conditions and times. Many fungal genera were gradually decreasing during storage and were eventually undetectable, and many fungal genera that were undetectable at first gradually increased during storage and even became dominant fungal genera. The competition between fungi was fierce. The competition between fungi were affected by the presence of DON. As the initial DON content increased, the contribution of inter-group differences became more obvious. The temperature, humidity, and CO2 concentration of wheat in the silo's environment changed with extended storage time. The content of DON decreased with extended storage time. We had investigated the changes in DON content and their correlation with the changes in fungal communities and environmental factors, which showed a high degree of correlation. This study offers theoretical justification for optimizing safe wheat grain in traditional storage conditions.