Fusarium head blight (FHB) is consistently one of the most important barley diseases worldwide. This study aimed to evaluate the pathogenicity of 16 isolates of four Fusarium species under controlled conditions and their genetic variability using 22 random amplified polymorphic DNA (RAPD) markers. Pathogenic variation was characterized based on disease development rates and disease index on two Syrian barley landraces with varying resistance to FHB, Arabi Aswad (AS) and Arabi Abiad (AB). Significant differences in intra- and inter-Fusarium species pathogenicity and in susceptibility between the above-mentioned cultivars were highlighted. Overall, the two barley landraces showed moderately susceptible to moderately resistance levels to fungal infection and FHB spread within the head. Quantitative traits showed significant correlation with previous data generated in vitro and under field conditions, suggesting that growth chamber indices can predict fungal pathogenicity and quantitative disease resistance generated under various experimental conditions. Based on PCR amplification with seven different primers, the isolates showed genetic variation. Dendrogram generated by cluster analysis based on RAPD markers data showed two main groups, suggesting that a possible clonal origin could exist in the four Fusarium species. RAPD fingerprints are not useful to distinguish the 16 Fusarium isolates with different levels of pathogenicity.