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The present study was performed to evaluate the effect of crop rotation on Fusarium mycotoxins and Fusarium species in cereals in Sichuan Province. A total of 311 cereal samples were randomly collected from 2018 to 2019 in Sichuan Province and analysed. The results of mycotoxin analysis showed that the major trichothecene mycotoxins in Sichuan Province were nivalenol (NIV) and deoxynivalenol (DON), and the mean concentration of total trichothecenes (including NIV, fusarenone X (4ANIV), DON, 3-acetyldeoxynivalenol (3ADON), and 15-acetyldeoxynivalenol (15ADON)) in wheat was significantly higher than that in maize and rice. The concentration of total trichothecenes in the succeeding crops was significantly higher than that in the previous crops. In addition, wheat grown after maize had reduced incidence and concentration of trichothecene mycotoxins compared with that grown after rice, and ratooning rice grown after rice had increased incidence and concentration of trichothecene mycotoxins. Our data indicated that Fusarium asiaticum with the NIV chemotype was predominant in wheat and rice samples, while the number of the NIV chemotypes of F. asiaticum and Fusarium meridionale, and the 15ADON chemotype of Fusarium graminearum in maize were almost the same. Although the composition of Fusarium species was affected by crop rotations, there were no difference when comparing the same crop rotation except for the maize–wheat rotation. Moreover, the same species and chemotype of Fusarium strains originated from different crops in various rotations, but there were no significant differences in pathogenicity in wheat and rice. These results contribute to the knowledge of the effect of crop rotation on Fusarium mycotoxins and Fusarium species affecting cereals in Sichuan Province, which may lead to improved strategies for control of Fusarium mycotoxins and fungal disease in China.