DNA sequence analysis of the alkaline protease gene was used to investigate two Aspergillus fumigatus strains isolated from ostriches (QLD1 and NSW3) and one environmental isolate (FRR 1266) that have shown genetic variation in previous analyses. The results showed that the QLD1 sequence was virtually identical to the published sequences for three human isolates but NSW3 differed in > 6% and FRR 1266 in > 10% of the nucleotides that were analysed. An RFLP assay was designed to determine the distribution of these (and other) genetic variants among environmental and clinical isolates of A. fumigatus.