In present investigation, vegetative compatibility groups (VCGs) of39 isolates of Fusarium graminearum collected from four provinces(Golestan, Mazandaran, Kerman, and Hormozgan) were determined using nitrate non-utilizing(nit) mutants. Results showed that with the exception of a single isolate (Fg-196)all the remaining isolates were forced to produce nit mutants on MMC and PDC media containing potassium chlorate. In total, 375 nit mutants were obtained of which, 72.5% were synthesized on MMC and 27.5% on PDC medium. On the basis of colony characteristics on basal medium containing one of the five nitrogen sources (sodium nitrate, sodium nitrite, ammonium tartarate, uric acid, and hypoxanthine) three nit mutant phenotypic classes were determined. Of these, 33.2% belonged to nit I, 28.2% to nit3, and 38.6% to nit M. To determine VCGs and complementation tests, pairing were made between all nit M and nit 1and I or nit3. In a compatible reaction between nit M and two other nit mutants, a dense growth at the line of contact between two colonies was formed after 5-14 days. But a much weaker similar phenomenon taking more than 14 days was observed, when nit 1and nit3 mutants were paired. All isolates examined in this study, were grouped nito 24 VCGs of which 14isolates, all from Goleston and Mazandarn provinces belonged to VCG 1, two isolates to VCG2 and each of the 22 remaining isolates (with various geographic origins) were classified in a separate VCGs. None of 39 isolates tested exhibited vegetative self-incompatibility reaction. In addition, no correlation was found between VCG2 and relative virulecnce of isolates.