Fusarium head blight (FHB) is recognized as a major threat to barley and oat production in Canada. The disease is caused by different species belonging to the genus Fusarium and leads to the accumulation of mycotoxins in grains that are toxic for humans and animals. Increases of FHB in barley and oat caused by infections with F. poae (FP), F. sporotrichioides (FS) and F. avenaceum (FA) have been observed in recent years. These pathogens produce Nivalenol, T-2/HT-2, and Enniatins, respectively, and are now considered important threats to the barley and oat industry. The impact of FP, FS and FA on barley and oat production has not yet been identified, and little is known about host resistance elements against these pathogens. This study investigated the pathogenicity of four Fusarium species, including F. graminearum (FG), FP, FS and FA, on ten barley and ten oat genotypes in 2019 and 2020. The levels of Fusarium mycotoxins, including deoxynivalenol, nivalenol, H-2/HT-2 and enniatins, in grain samples were analyzed using an LC-MS/MS method. All Fusarium species tested caused infection and mycotoxin contamination in different barley and oat genotypes. The resistance in barley and oat genotypes against FG and FA shows a similar pattern but differs from the responses against FP and FS. Overall, the level of DON in barley and oat grain was the highest, ranging from 0.1 to 5.8 ppm for barley and from 0.1 to 11.5 ppm for oat. NIV had the lowest concentrations in 2019 and 2020, ranging from 0.01 to 0.36 ppm in barley and from 0.01 to 0.16 ppm in oat.