Understanding the effects of temperature on Fusarium graminearum infection can provide theoretical guidance for chemical control of Fusarium head blight (FHB). Here, we evaluated the effects of various temperatures on biological fitness development of wild-type sensitive strain 2021 and carbendazim-resistance mutants conferring β₂-tubulin substitutions F167Y, E198K, and E198L. The results showed that mycelial growth and conidiation of four strains increased with the increase in temperature between 10 and 25°C. Conidia of F167Y displayed strong adaptability to low temperature. The virulence of the four strains was largely similar at the same temperature, showing an upward trend between 10 and 25°C. At 10°C, the hyphal growth of all strains was significantly inhibited, metabolism was slowed down, and the accumulation of secondary metabolites decreased. Subsequently, the production of deoxynivalenol (DON) and its intermediates pyruvate and aurofusarin decreased at low temperature, and the expression of DON biosynthesis-related genes Tri5, FgPK, and AUR decreased accordingly. At the same temperature, the aurofusarin production of the strains F167Y and E198K was higher than that of strains 2021 and E198L. The contents of DON and pyruvic acid in carbendazim-resistance mutants were higher than those in the wild-type strain 2021. The sensitivity of four strains to different fungicides changed at various temperatures. The sensitivity to most fungicides increased with decreasing temperature. The carbendazim-resistance mutants showed positive cross-resistance with other benzimidazoles. However, there was no cross-resistance to pyraclostrobin and azoles. These results would direct us to use fungicides preventing the infection of F. graminearum with changeable atmospheric temperature at the wheat flower stage.