This study aimed to investigate the mycoflora and chemotyping of Fusarium graminearum isolates in corn and barley used in industrial dairy farms in Khorasan-Razavi Province, northeastern Iran. For this purpose, a total of 128 corn and barley samples were collected from 16 different large industrial dairy farms. Mean moisture of the samples collected in different seasons of the year was significantly different (P < 0.05). The most prevalent fungi isolated from corn samples were Penicillium spp. (90.62%) and Aspergillus spp. (82.81%), and that of barley samples was Penicillium spp. (89.06%). Frequency of Fusarium spp. İn corn and barley samples was 85.93% and 26.56%, respectively. All isolates (100%) were confirmed as Fusarium using an ITS primer. Eight (11.11%) out of 72 Fusarium spp. İsolates were confirmed as F. Graminearum using species-specific primers. Five out of 8 F. Graminearum isolates were positive in terms of the presence of the trichothecene-producing gene using a Tri5-specific primer. Three and 2 out of 5 toxin-producing F. Graminearum isolates were positive using Tri13DON- and Tri13NIV-specific primers, respectively. In conclusion, the presence of toxigenic Fusarium in corn and barley samples used in feedstuffs in Iran is a potential risk to animal and human health.