The Current study revealed the natural occurrence of toxigenic fungi and mycotoxins production in grains in Saudi Arabia. Samples of yellow corn, white rice and red corn grains were collected from different local markets. Three fungal isolates were isolated from the examined corn grains using PDA media. The toxigenic Alternaria spp. was the most prominent fungi in yellow corn grains, white rice and red corn grains. Three Alternaria spp. isolated were identified using molecular characterization of ACTTS gene. DNA genome of the three Alternaria spp isolates (namely AWR; AYC and ARC which corresponds to isolates from white rice, yellow corn, red corn) was used as a template for PCR to amplify ACTTS gene. Partially sequenced ACTTS gene was amplified using a specific primer set to confirm its identity, phylogenetic relationships between the three isolates as well as determination of the corresponding antigenic determinants. The epitope prediction analysis demonstrated that there were 5, 6 and 5 epitopes whose score were above 0.90 in AWR, AYC and ARC, respectively. Interestingly, there were great variations in the epitope sequences among the three isolates except for the epitope, VYGASTATGTLAVQ. This work led to molecular identification of three Alternaria spp. using ACTTS gene and the unique antigenic determinants that could be used for design of a broad spectrum antibody for rapid detection of Alternaria spp. in foods.