Fusarium proliferatum is the causal agent of stalk and root rot disease of maize, foot rot disease of rice, basal and root rot disease of onion and knife cut disease of sugarcane in Iran. In recent years, incidence and severity of these diseases have been increased in Iran. Fifty seven F. proliferatum single-spore isolates collected from diseased maize, rice, onion and sugarcane plants at different areas were used to study genetic diversity by determination of vegetative compatibility groups (VCGs). Chlorate-resistant nitrate non-utilizing (nit) mutants were recovered from selected isolates of F. proliferatum and used in complementation tests. All isolates in which both nit1 and NitM (or nit3) mutants were recovered, demonstrated self-compatibility. Vegetative compatibility tests by pairing nit mutants identified 30 VCGs among 57 isolates. Twenty-three isolates belonged to singlemember VCGs and the remaining 34 isolates, belonged to other seven multimember VCGs. Segregation of F. proliferatum isolates obtained from various area and host plants into different VCGs in Iran is reported for the first time. In this study, none of isolates obtained from rice complemented with any other isolates from onion and sugarcane and, non complementation occurred between onion and sugarcane isolates. Also, only one complementation occurred between one isolate of maize and one isolate of sugarcane and rice. Thus, a correlation between VCGs grouping and host preferences was founded. It is concluded that natural populations of F. proliferatum in Iran are probably genetically divergent and include isolates representing a potential risk for disease development.