Fusarium culmorum and F. graminearum are the major pathogens for dryland root/foot-rot and head-blight diseases in economically important grain crops. This study was aimed at the molecular characterization of Fusarium spp. isolates, which have been collected from cereal fields in three agro-ecological regions in Turkey. Genetic diversity has been analyzed by generating RFLP markers from the intergenic spacer (IGS) region of ribosomal RNA. The selection of restriction enzymes for IGS-RFLP studies has been found critical to maximize polymorphic markers. Only 3 of 14 restriction endonucleases were useful in differentiating Fusarium spp. isolates. PstI was the most efficient enzyme to produce a maximum of nine DNA markers in one individual and total 22 polymorphic representative banding patterns. Polymorphism based on IGS-RFLP was high and average 88% in both species. There was no association between IGS diversity and geographic locations from which the samples were taken. Both MAT-1 and MAT-2 sequences were amplified in F. graminearum similarly to previous reports. Most of the F. culmorum isolates carried either MAT-1 or MAT-2 sequences, and differently two isolates carried both sequences. Mating type determination was helpful to distinguish F. pseudograminearum from F. graminearum, which cannot be discriminated by SCAR markers or morphological assessment. High genetic diversity by IGS-RFLP markers in F. culmorum was discussed in relation to its fitness as the most common pathogen in dryland root rot complex (DLRRC).