Thirty one isolates belonging to six fungal genera were found to be associated with wheat grain samples collected from three main regions in Saudi Arabia. The most common genera (average frequency) were Aspergillus (19.52%), Fusarium (32.31%), Penicillium (12.19%), and Alternaria (8.2%). In this study, different isolates of Fusarium spp. were isolated from wheat grains samples and identified at the molecular level by ITS-rDNA regions amplification. Twenty five isolates of Fusarium spp. were screened for their ability to produce Fumonisin B1 (FB1), nivalenol (NIV) and deoxynivalenol (DON) using HPLC. Eight isolates were capable of producing detectable levels of FB1, NIV and DON. Inter-simple sequence repeats (ISSR) molecular markers were used, with the aim of genetically characterizing isolates of Fusarium spp. and discriminate between atoxigenic and toxigenic isolates. ISSR analysis revealed a high level of genetic diversity in the Fusarium spp. population and useful for genetic characterization. ISSR markers were not suitable to discriminate atoxigenic and toxigenic isolates also, Clustering based on ISSR dendrograms was unrelated to geographic origin.