Aflatoxins (AFs) are toxic and carcinogenic secondary metabolites produced by isolates of Aspergillus section Flavi as well as a number of Aspergillus isolates that are classified outside of section Flavi. Characterization of the AF and sterigmatocystin (ST) gene clusters and analysis of factors governing regulation of their biosynthesis has resulted in these two mycotoxins being the most extensively studied of fungal secondary metabolites. This wealth of information has allowed the determination of the molecular basis for non-production of AF in natural isolates of A. flavus and domesticated strains of A. oryzae. This review provides an overview of the molecular analysis of the AF and ST gene clusters as well as new information on an AF gene cluster identified in the non-section Flavi isolate, Aspergillus ochraceoroseus. Additionally, molecular phylogenetic analysis using AF biosynthetic gene sequences as well as ribosomal DNA internal transcribed spacer (ITS) sequences between various section Flavi and non-section Flavi species has enabled determination of the probable evolutionary history of the AF and ST gene clusters. A model for the evolution of the AF and ST gene clusters as well as possible biological roles for AF are discussed.