Members of the genus Aspergillus are opportunistic fungal pathogens characterized by their genomic diversity. However, whether variations among Aspergillus strains and species at the genome level translate into significant differences in virulence is unclear. Therefore, we studied the interstrain and interspecies variations in virulence for a collection of Aspergillus fumigatus and Aspergillus terreus isolates using a previously described model of invasive aspergillosis in Toll-deficient fruit flies. We then looked for associations between survival in the fly model and strain relatedness as defined by repetitive-sequence polymerase chain reaction (rep-PCR). We observed no significant differences in the survival of flies infected with A. fumigatus vs. A. terreus or flies infected with colonizing vs. invasive isolates of either species. However, in both Aspergillus species we observed significant interstrain variability in fly survival (P<0.001 by the log-rank test). Using rep-PCR, we identified two dominant A. fumigatus clades that were associated with significantly different survival rates in Toll-deficient flies (P=0.007). We conclude that the fly model of invasive aspergillosis enables high-throughput screening of Aspergillus species for variations in virulence and may uncover distinct A. fumigatus clades that differ in their pathogenicity.