Aspergillus flavus is one of the most abundant and widely distributed fungi on earth. A. flavus produces aflatoxins (AFs), which are toxic secondary metabolites. AFs have harmful effects on public health (humans and animals) and agricultural crops. Inter-simple sequence repeat (ISSR) markers were used to analyze the genetic diversity of 30 A. flavus isolates from five agricultural crops and air. Genetic similarity coefficients (GSC) ranged from 0.51 to 0.10 based on three ISSR markers for the isolates tested. A. flavus isolates grouped into 6, 5, and 3 clusters using the unweighted pair-group method with arithmetic average of three ISSR markers. This study suggests that ISSR biotechnology is a highly useful tool for characterizing genetic diversity of A. flavus isolated from different sources.