Diversity of trichothecene chemotypes of Fusarium graminearum isolated from kernels of wheat, barley and maize grown under various agro-ecological conditions on 13 locations was analysed. Sixteen strains were tested for the effective capability to produce 15-ADON, 3-ADON and NIV, by using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Fourteen out of sixteen analyzed strains produced 15-ADON, while remaining two were of the 3-ADON chemotype. Multiplex PCR reaction with two sets of specific primers for TRI3 and TRI12 genes was applied to identify trichothecene chemotypes (3-ADON, 15-ADON and NIV). The expected sizes of amplified fragments for TRI3 gene primer set are 840 bp (NIV), 610 bp (15-ADON) and 243 bp (3-ADON). The amplified fragments for TRI12 gene primer set should be 840 bp (NIV), 670 bp (15-ADON) and 410 bp (3-ADON). All F. graminearum isolates were of the 15-ADON chemotype, i.e. their bands were 610 bp and 670 bp size for TRI3 and TRI12 genes, respectively. The results indicate that genotypic characterisation does not correspond to determined chemotypes and this is a reason why the analyses for the risk of mycotoxins contamination should not be based only on trichotecene genotype determination. Due to high temperature differences in cereal growing regions in Serbia, the presence of other chemotypes could be expected. In order to determine whether besides 15-ADON there are other F. graminearum chemotypes on wheat, barley and agro-ecological conditions.