Abstract: The tri5 gene cluster is associated with production of trichothecene B type mycotoxins in Fusarium graminearum and F. culmorum. Multifunctional oxygenase and trichodiene synthase encoded by tri4 and tri5 genes, respectively, belonging to this cluster are responsible for production of trichothecenes. In this study, tri4 and tri5 gene expression levels were determined in 14 F. graminearum and 24 F. culmorum isolates causing head blight in wheat, maize and barley collected from different regions of Turkey. Isolates producing oxygenase and trichodiene synthase in high levels were distinguished from low-producers using two-step reverse-transcription comparative quantitative real-time PCR. Slope values were -3.63, -3.4 and -3.4 for amplification of tri4, tri5 and β-tubulin genes, respectively. Cp values were ranged from 17.08±0.0 to 38.82±1.66. The highest tri4 and tri5 expression levels were determined in 4F isolate of F. graminearum. ΔΔCT ratios were calculated as 0.1371 for tri4 gene and 0.865 for tri5 gene. However, maximum tri4 and tri5 expressions were detected in 20F (ΔΔCT = 0.2196) and 9F (ΔΔCT = 0.3761) isolates in F. culmorum, respectively. Quantification of definite toxin content can contribute to determination of pathogen infection grade and to gaining the information about fungal biomass on field crops and their products.