BACKGROUND: Mycotoxin produced by mould is one of the most serious contamination sources in food security. Safe storage of grain has become more important to control food security. Currently, there is no officially approved or standardized sampling scheme for detecting mycotoxin in grain storage worldwide.RESULTS: In this study, deoxynivalenol (DON) was taken as a typical mycotoxin in stored wheat to be detected. Population density of corn weevil could not significantly increase wheat moisture, but wheat moisture was highly significantly and positively correlated with DON content (P < 0.01). Corn weevil density significantly increased the DON content in wheat. DON contamination degree was mainly distributed in the region of 14-20 cm below the surface layer of wheat. In the process of ventilation and dehumidification during the storage period, moisture of wheat decreased slightly with the extension of ventilation, but the DON content in wheat increased significantly. Combined with the analysis of ventilation, DON content in the upper layer and H1 position, where the wind direction is not easy to reach, increased significantly. CONCLUSION: Areas with high insect population density (14-20 cm below the surface layer of stored wheat) and low ventilation and high humidification (H1 position in the upper layer) should be taken as the key cutting sample areas for detecting mycotoxin during the period of grain storage. This study provides for the first time a scientific basis for the standardization of the wheat sampling scheme to monitor mycotoxin contamination during wheat storage.