Barley is the third most important cereal crop in terms of production in Canada, and Fusarium head blight (FHB) is one of the main fungal diseases of barley. FHB is caused by a species complex of Fusaria, of which Fusarium graminearum Schwabe is the main causal species of FHB epidemics in Canada. Field surveys show that two or more Fusarium species often co-exist within the same field or grain sample, and F. poae is reported as another important species in barley. This study aimed to determine the pathogenicity of F. graminearum, F. poae, and a co-inoculation of both species causing FHB in barley. Two susceptible barley cultivars were spray-inoculated at 10 to 14 days after heading. Phenotypic disease severity was rated on a scale of 0-9 at 4, 7, 14, 21, and 28 days after inoculation. There was a significant difference in FHB severity between F. graminearum and F. poae, where infection with F. graminearum produced more severe disease ratings. F. poae generated lower disease ratings and was not statistically different from the control. When heads were co-inoculated with both Fusarium species, the resulting FHB severity was unchanged relative to heads inoculated with F. graminearum only. The ratio of F. graminearum to F. poae genomic DNA was also no different than when heads were inoculated with F. graminearum alone, as quantified with ddPCR using markers specific to each species. The metabolomic analysis of sample extracts showed that F. graminearum-associated metabolites dominated the mycotoxin profile of co-inoculated samples, which corroborated our other findings where F. graminearum appeared to outcompete F. poae in barley. No significant effect on visual FHB disease ratings or fungal DNA detection was observed between the cultivars tested. However, there were some metabolome differences between cultivars in response to the challenge by both F. graminearum and F. poae.