In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB1, FB2, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rDNA. The results indicated that G. fujikuroi complex isolates can be divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.