Environmental isolates of Aspergillus fumigatus are less studied than those recovered from clinical sources. In the present study, the genetic diversity among such environmental isolates was assessed, as well as their dispersion ability and the acquisition of new strains in 19 medical units of the same hospital. A. fumigatus isolates were genotyped using a single multiplex PCR-based reaction with eight microsatellite markers and an insertion/deletion polymorphism. A total of 130 unique genotypes were found among a total of 250 A. fumigatus isolates. Genotypic diversity ranged from 0.86 to 1 in samples from hospital rooms, and there was no correlation between these samples and the presence of high-efficiency particulate air filters or any other air filtration system. Four of the six most prevalent A. fumigatus strains were recovered from water samples. The occurrence of microvariation was common among environmental isolates, which affected each of the microsatellite markers. The assessment of the genetic diversity of A. fumigatus is a useful tool for illustrating the presence or absence of specific clonal populations in a clinical setting. A. fumigatus populations were highly dynamic indoors, and new populations were found in just a few months. Due to the high indoor dispersion capability of A. fumigatus, more attention should be given to strains with increased pathogenic potential or reduced susceptibility to anti-fungal drugs.