Barley is one of the most important cereals consumed in the world, which is well-known to be invaded by Aspergillus spp. under storage conditions and contaminated with mycotoxins. Aflatoxin (AFL) contamination of cereal grains especially barley is one of the serious economic and food security issues. This study aimed to assess the mycological and AFL contamination of barley collected from market in Kermanshah province, in west of Iran. Forty samples were collected at informal markets in ten cities and analysed for aflatoxin B₁ using enzyme-linked immune sorbent assay (ELIZA) method. Sixty Aspergillus isolates were collected and four species were identified. All these strains belonged to A. niger (50%), A. flavus (30%), A. japonicus (15%), and A. sclerotiorum (5%). Further confirmation of all the A. flavus isolates was carried out on molecular basis through species specific primers of AFLA-F/AFLA-R. Natural occurrence of AFB₁ was detected in 18 samples with a range between 0.056–10.564 μg kg^-1. The highest AFB₁ level was found in samples from Sarpol-e-Zahab (10.564 μg kg^-1). This is the first report of the natural occurrence of AFB1 from stored barley samples collected from west of Iran.