Fusarium Head Blight is the most important disease of cereals worldwide. Studies of interactions between different flower/grain pathogenic Fusarium species have been carried out, using the host and tissue from which the fungus was isolated. The aim of the present study was to assess the root responses of Brachypodium distachyon genotypes to Fusarium spp. to provide information about differences in host resistance and/or fungal aggressiveness. A total of 10 isolates of four Fusarium species were selected: F. graminearum, F. pseudograminearum, F. cerealis, and F. poae. A total of seven genotypes of B. distachyon were used: Afghanistan, Iran, Israel, Pakistan, South Africa, Uruguay and Bd genotype 21. Roots were inoculated and the necrosis root length was measured at 2, 4, 6, and 8 days post-inoculation. The results showed that F. graminearum and F. pseudograminearum were the species with the highest AUDPC values followed by F. cerealis that showed an intermediate value of AUDPC. F. poae was the least aggressive species. Regarding Brachypodium, the least affected genotype was Bd 21 and Pakistan, while the remaining genotypes showed differences in susceptibility. To our knowledge, this is the first report showing interaction between F. pseudograminearum, F. cerealis and F. poae with Brachypodium roots. Moreover, variability in susceptibility among Brachypodium genotypes and in Fusarium species aggressiveness was reported. We concluded that B. distachyon roots offer an efficient tissue system model to evaluate interaction with Fusarium spp.