Abstract: The sensitivity of 100 Fusarium graminearum isolates from Jiangsu, Anhui, Shandong and Henan Province in China during 2012-2014 to metconazole was determined through the mycelial growth rate method, metconazole-resistant mutants were obtained through spontaneous selection and the fitness and sequences, expression level of the CYP51 gene of the resistant mutants were analyzed. The EC₅₀ value of 100 isoaltes to metconazole ranged from 0.04-0.51 μg/mL and with an average value of (0.18±0.09) μg/mL. The sensitivity frequency of F. graminearum to metconazole distributed as a unimodal peak curve and no resistant sub-population was found. This average EC₅₀ value could be used as baseline-sensitivity for field resistance monitoring. Twelve metconazole-resistant mutants were generated through spontaneous selection. Among them, two mutants displayed moderate-resistance with resistance factor (RI) ranged 14.2 and 15.8 and other ten mutants displayed low-resistance with RI ranged from 3.25 to 9.05. Compared with the parent isolates, the mycelial growth and conidia production ability of some resistant mutants decreased significantly and the pathogenicity of all resistant mutants decreased significantly. Cross resistance studies showed that some metconazole-resistant mutants also showed resistance to tebuconazole, propiconazole and prochloraz, but not to prothioconazole and triadimefon. Some metconazole-resistant mutants showed resistance only to tebuconazole, but not to propiconazole, prochloraz, prothioconazole and triadimefon. And all metconazole-resistant mutants were sensitive to phenamacril. Study has showed that an overall low risk of resistance development in F. graminearum was recommended for metconazole. Compared with the parent isolate, no mutation in CYP51 genes and their promoter regions were found in two moderate-resistant mutants and two low-resistant mutants and CYP51A gene expression was all up-regulated in four mutants with 1.33-10.28 times. So, we concluded that CYP51A gene expression might be related to the resistance to metconazole in F. graminearum.