In recent years, plant breeders made great progress in breeding Fusarium-tolerant wheat lines. However, total resistance to this genus of plant pathogenic fungi has not yet been achieved as the resistance genes are located on several distinct genetic regions. Visual scoring of disease symptoms in combination with the analysis of mycotoxins is commonly applied to assess the tolerance of new lines. Both approaches are indirect methods and do not mandatorily determine the accumulated fungal biomass. Quantitative PCR is a useful tool to assess fungal biomass based on the abundance of organism-specific DNA. The aim of this study was the development of a quantitative PCR assay for trichothecene-producing Fusarium species and to adapt this method for resistance assessment of wheat lines artificially infected with Fusarium graminearum and Fusarium culmorum. Several DNA-extraction methods for wheat samples were evaluated and optimized for downstream real-time PCR analysis and furthermore, a new reference-gene-based approach for more accurate quantification of Fusarium biomass in cereals is presented. The co-determination of a plant gene was used to compensate for unequal DNA-extraction efficiencies.