Microsatellite analysis was used to examine the genetic relatedness of 111 clinical and environmental isolates of the opportunist human pathogenic fungus Aspergillus fumigatus from Ontario, Canada. Forty-three A. fumigatus isolates were from clinical sources and 68 from environmental sources. Phylogenetic analysis of the genotypes revealed that there were no geographical or temporal associations of clinical or environmental genotypes. In fact, several of the environmental and clinical isolates showed identical (clonal) genotypes from disparate geographical areas. However, a locus by locus examination revealed that there were several significant differences in allele frequencies between clinical and environmental isolates. There may be linkage of certain microsatellite loci with genes affecting virulence in A. fumigatus. A susceptible individual may be equally predisposed to infection by any isolate of A. fumigatus. However, under transient selection as a pathogen, genes encoding alleles for enhanced virulence may not assort independently from microsatellite loci. A dynamic equilibrium may exist between random recombination of loci in the natural environment and selection for virulence factors during host infection cycles.