Thirty samples consisting of wheat (15) and barley (15) were collected from different markets in Penang, Malaysia, originating from India and Thailand, respectively. All samples were analyzed for occurrence of Aspergillus spp. and aflatoxin B1 (AFB1). Aspergillus flavus was dominant in all samples followed by A. niger. AFB1 could be detected in three wheat samples ranging from 0.42 to 1.89 μg/kg and one barley sample had 0.58 μg/kg of AFB1. The AFB1 levels in all the samples were below the Malaysian regulatory limits (<35 μg/kg). The frequency and quantity of AFB1 levels in this study were very low in wheat and barley samples compared to other agricultural commodities reported in India and Thailand. This is the first report on determination of Aspergillus spp. and AFB1 in imported wheat and barley grains in Penang, Malaysia.