Fungi mycotoxins can be a serious risk to health and lead to substantial economic loss. The environmental conditions of Saudi Arabia, with its mostly warm temperatures, are conducive to the growth of toxigenic fungi resulting in mycotoxin production in different food items. The current study elucidates the natural occurrence of toxigenic fungi and mycotoxin production in grains in Saudi Arabia. Samples of white rice and corn (yellow, red) grains were collected from different local markets and houses. Three fungal isolates were obtained from the corn and rice grains and examined using Potato Dextrose Agar (PDA) and Carnation Leaf Agar (CLA) media. Fusarium spp. were the most prominent fungi in yellow corn, red corn and white rice grains. Three isolated F. moniliforme strains were identified using molecular characterization of the trichothecene 3-O acetyltransferase (TRI101) toxin gene. The DNA genome of the three Fusarium moniliforme isolates (namely, F. moniliforme_1, F. moniliforme_2 and F. moniliforme_3, which correspond to isolates from yellow corn, red corn and white rice, respectively) were used as a template for PCR to amplify trichothecene 3-O acetyltransferase (TRI101). Partially sequenced fragments amplified using a specific primer set were used to confirm the identification of, and to evaluate the phylogenetic relationships among the three isolates as well as to identify the corresponding antigenic determinants. The epitope prediction analysis demonstrated that there were four epitopes with scores equal to 1 in F. moniliforme_1, F. moniliforme_2 and F. moniliforme_3, respectively. Interestingly, there were great dissimilarities in the epitope sequences among the three isolates except in NSTPRACASEQEVS, STSSRADSSSLSTD and CTLCPRSLMASSVR. This indicates that the unique antigenic determinants predicted in the trichothecene 3-O acetyltransferase (TRI101) toxin gene could be used for designing a broad spectrum antibody for rapid detection of Fusarium spp. in foods.