Numerous patients were diagnosed with aspergillosis in a nosocomial outbreak caused by Aspergillus fumigatus and Aspergillus flavus. Thirty-three isolates of the former and 28 isolates of the latter were collected from the hospital environment and from the patients and studied for genetic relatedness by random amplified microsatellites (RAMS) analysis, in which two polymorphic regions were tested. Twenty-eight genotypes of A. fumigatus and 23 genotypes of A. flavus were identified. Four patients were infected by two isolates with the same genotype as the environmental isolates. One clinical genotype was shared by three patients and another was shared by two patients. We found that RAMS was useful for fingerprinting Aspergillus spp.