Fungi belonging to Aspergillus section Flavi are of great economic importance in the United States due to their ability to produce toxic and carcinogenic aflatoxins in agricultural commodities. Development of control strategies against A. flavus and A. parasiticus, the major aflatoxin-producing species, is dependent upon a basic understanding of their diversity in agricultural ecosystems. This review summarizes our current knowledge of species and population diversity in the United States in relation to morphology, mycotoxin production and genetic characters. The high genetic diversity in populations of aflatoxigenic fungi is a reflection of their versatile habits in nature, which include saprotrophic colonization of plant debris in soil and parasitism of seeds and grain. Genetic variation within populations may originate from a cryptic sexual state. The advent of intensive monoculture agriculture not only increases population size but also may introduce positive selective pressure for aflatoxin production due to its link with pathogenicity in crops. Important goals in population research are to determine how section Flavi diversity in agricultural ecosystems is changing and to measure the direction of this evolution.