Azole fungicides play a prominent role for reliable plant disease management. However, quantitative azole resistance has been shown to develop in fungal pathogens, including Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Due to widespread application of azole fungicides, resistance may accumulate to higher degrees in fungal field populations over time. Although azole fungicides are prominent components in FHB control, little effort has been made to investigate azole resistance in F. graminearum. We allowed F. graminearum strain NRRL 13383 to adapt to an azole fungicide in vitro, applying a strongly growth-reducing but sublethal dose of tebuconazole. Two morphologically distinguishable azole-resistant phenotypes were recovered that differed with regard to levels of fitness, fungicide resistance, virulence, and mycotoxin production. Isolates of the adapted "phenotype 1" exhibited azole-specific cross-resistance, whereas "phenotype 2" isolates displayed the phenomenon of multidrug resistance because the sensitivity to amine fungicides was also affected. Assessment of individual infected spikelets for mycotoxin contents by high-performance liquid chromatography mass spectrometry and for Fusarium DNA by quantitative polymerase chain reaction indicated that some of the adapted isolates produced significantly higher levels of nivalenol per fungal biomass than the NRRL 13383 strain.