Fusarium head blight (FHB) is an important cereal disease and may result in the accumulation of toxins in grains. Although F. graminearum is globally the most prevalent FHB causing species, F. avenaceum is found in contaminated samples. Real-time PCR (qPCR) is the the standard analytical method for species-specific, quantitative estimation of fungal biomass in the tissue of host organisms. qPCR is useful for quantifying fungal colonization of crops while distinguishing among species. Recently, species-specific PCR primers have been developed for most Fusarium species that cause head blight. In some cases, the species-specific primers synthesized with other Fusarium species and can give the wrong results. In this study, two species-specific primer pairs for F. avenaceum were tested by using with different fungi DNA. JIA primer pairs were amplified only F. avenaceum, whereas MGA primer pairs were amplified with F. equiseti and F. tricinctum. Results indicate that primer pair JIA is effective in determination of F. avenaceum.