Aspergillus flavus and A. parasiticus are closely related species belonging to the Aspergillus section Flavi. Both species can produce aflatoxins, but not all isolates of either species do so. We examined the genetic relationship between toxigenic and non-toxigenic isolates using RAPD and Neighbour Joining analysis. Twenty-four RAPD amplifications using a combination of 17 primers discriminated between 20 isolates of A. flavus and 15 isolates of A. parasiticus. A. flavus isolates divided into two distinct groups with both toxigenic and non-toxigenic isolates occurring in each group. No association was seen between RAPD genotype and the ability to produce toxin. Five non-toxigenic isolates of A. parasiticus separated into two groups, in which the isolates were similar but not identical. These groups of non-toxigenic isolates occurred on branches in which toxigenic isolates also occurred. This study suggests that either multiple losses of toxigenicity in A. flavus and A. parasiticus have occurred, or that recombination has reassorted this phenotype into a variety of different genetic backgrounds.