Aim of this study was to develop sensitive PCR assay for mycotoxin producing Fusarium species. Strains Fusarium oxysporum 4199 and Fusarium culmorum 4044 were used as representatives of this group. Primers JB chosen to demonstrate the affiliation to genus Fusarium were derived from ITS region of rDNA. Gene from trichothecene pathway Tri4 was employed to design primers for toxin biosynthesis. Specificity of PCR based on JB primers was tested on DNA isolated from F. culmorum 4044, F. oxysporum 4199, Aspergillus oryzae 4002 and Mucor circinelloides 4018, Trichoderma sp. Both Fusarium species gave positive reaction, while the later ones did not react. Primers based on Tri4 highly specific sequences were giving positive reaction only with DNA from F. culmorum 4044 and F. oxysporum 4199. DNA isolated from six samples of contaminated wheat grains gave positive result on the presence of genus Fusarium and mycotoxines by optimised PCR protocol using JB and Tri4 primers. The results corresponded to LC/MS analysis that was established quantitatively in all samples to ascertain the amount and type of fusarious mycotoxines.