The real-time PCR-based system has been developed for the detection and quantification of the most common Fusarium species with the similar profiles of mycotoxins they can potentially produce. Corresponding group-specific primers for various Fusarium fungi have been designed based on the translation elongation factor 1α (tef1α) gene sequences. The specificity of the selected primers and TaqMan probes has been tested with 26 single-spore isolates of seven most common Fusarium species and 21 wheat and barley grain samples. PCR results were analyzed relative to the most common mycotoxin deoxynivalenol (DON) content in infected grain samples. © 2010 Elsevier Ltd.