Fusarium species belong to one of the most important fungal groups in the medical, agricultural, and veterinary fields. This study aimed to evaluate the efficiency of PCR-RFLP analysis of the beta (β)-tubulin region for differentiating Fusarium species. A total of 107 strains of Fusarium spp. were studied, including isolates from environmental, clinical, and reference sources. The β-tubulin genes of all isolates were successfully amplified with primer pairs (T1 and T22). A PCR product of approximately 1400 base pairs was generated for each Fusarium sp. After evaluation of various enzymes, three restriction enzymes, namely Ban II, BsaWI, and HincII, were selected. Based on the selected enzymes, the isolated Fusarium spp. were categorized into 24 groups. In this study we were able to identify F. graminearum, F. culmorum, and F. cerealis through the proposed analyses as well as other pathogenically important species such as F. oxysporum and F. solani. Unlike all other similar previous studies, this study was able to differentiate among F. graminearum, F. culmorum, and F. cerealis. However, we were unable to differentiate F. armeniacum and F. acuminatum or F. sportrichioides from F. langsethiae. Hence, it is recommended that other genes must be evaluated to overcome the limitations of the ?-tubulin gene in differentiating the above species.