Fusarium culmorum is a phytopathogenic fungus causing Fusarium head blight (FHB), which negatively affects cereals by producing mycotoxins, such as deoxynivalenol (DON). In this work, two barley cultivars, Chevron and Pedant, with different degrees of resistance to FHB were inoculated with F. culmorum. The transcription levels of the Fusarium Tri genes and barley UDP‐glycosyltransferase genes were investigated. The amounts of pathogen, DON and the detoxification product deoxynivalenol‐3‐O‐glucoside (D3G) were monitored. The greatest amounts of pathogen were detected at 21 days postinoculation (dpi) and were much lower in cv. Chevron than in cv. Pedant. No differences in the total DON conversion to D3G were observed between the cultivars. Ubiquitin‐conjugating enzyme (UBC) was identified and then used as a reference gene to monitor transcription of the Fusarium Tri genes in infected barley. Transcription of the F. culmorum Tri5, Tri4, Tri6 and Tri10 genes differed between the two cultivars. In the susceptible cultivar (Pedant), transcription of the Tri genes gradually increased from 1 dpi. In the more resistant Chevron, transcription of the Tri genes dramatically increased after 14 dpi and reached a maximum at 21 dpi. This very high but delayed transcription of Tri genes did not, however, result in a large accumulation of the mycotoxin DON. The difference between the cultivars in the transcription of barley defence genes (HvUGT13248 [GT2] and HvUGT5876 [GT1]) for UDP‐glycosyltransferases reflects the barley samples’ levels of infection. The difference in resistance to F. culmorum infection in the two cultivars is most likely not due to differences in DON detoxification, but may be due to activity against the pathogen and delayed transcription of the pathogen's Tri genes.