Aspergillus flavus is the second leading cause of allergic, invasive, and colonizing fungal diseases in humans, and also the second most frequent organism associated with avian infections. Currently, it is not known whether there is a link between the environmental isolates and/or human isolates of A. flavus and those responsible for aspergillosis in birds. Microsatellite typing was used to analyze 29 A. flavus clinical and environmental avian isolates and 63 human clinical isolates collected from patients with a variety of aspergillosis diseases. The combination of all six markers yielded 77 different genotypes with a 0.98 D value. A. flavus genotypes obtained from avian isolates were compared with those obtained from human clinical and environmental samples. The standardized indices of association I (A) and rBarD were significantly different from zero (p < 0.01), suggesting a prevailing clonal reproduction. There was high genetic diversity between the hospital and poultry environments of A. flavus isolates. The human environmental population was significantly differentiated from environmental and clinical avian populations (F (st) > 0.25). The avian clinical subpopulation exchanged few strains with the environmental human (N (m) = 7.24) and avian (N (m) = 6.60) populations. The minimum spanning tree analysis identified three A. flavus genotype clusters that were highly structured according to the isolation source (p < 10(-4)).