Isolates of Fusarium from rice, sugarcane and maize were identified as F. verticillioides, F. sacchari, F. proliferatum F. subglutinans, F. fujikuroi and F. oxysporum. The species were then characterized by restriction analysis of intergenic spacer (RFLP-IGS) using AluI, Eco88I, RsaI and XhoI. Twenty-five haplotypes were identified among the isolates of Fusarium which indicated high levels of variations. UPGMA cluster analysis was conducted to cluster the isolates and to estimate the intraspecific and interspecific variability. Isolates of F. fujikuroi from rice were clustered together with isolates of F. proliferatum from rice and maize with a similarity value of 88–100%. Isolates of F. verticilliodes from maize and sugarcane were clustered together with a similarity value ranging from 92–100%, and two isolates from rice formed another cluster. Isolates of F. oxysporum from maize and rice were clustered together with a similarity value ranging from 87–95%. Isolates of F. subglutinans from rice and maize, and F. sacchari from rice and sugarcane were also clustered together with a similarity value of 77–100%. Based on RFLP-IGS analysis, variability was observed within and between species of Fusarium from rice, maize and sugarcane and the technique could be use to complement morphological characterization and to determine genetic relationships between the species.