Aspergillus fumigatus is a known opportunistic pathogen of penguins. Most reports of penguin aspergillosis have focused on clinical, histopathological and microbiological findings. The molecular characterization of A. fumigatus isolates involved in invasive infections in penguins has yet to be addressed. The aim of this work was to study the possible coexistence of different A. fumigatus genotypes in five clinical cases of invasive aspergillosis in captive penguins. Differences in other relevant characteristics of the isolates, including mating type and invasiveness, were also considered. Alkaline protease and elastase production by the A. fumigatus isolates was evaluated by plate assays. Random amplified polymorphic DNA, and microsatellite analysis techniques were used for molecular typing, and mating type (MAT1-1 or MAT1-2) was determined by multiplex PCR. Although all isolates showed protease activity, differences in elastase activity were observed. The typing techniques indicated different genotypes in all the penguins, although one genotype was predominant in some cases. Fungal strains of different mating type were found in two different penguins, confirming infection polyclonality. In conclusion, captive penguins are susceptible to infection by multiple strains of A. fumigatus that differ not only in their genotype, but also in mating type and invasiveness. This finding has important consequences for the diagnosis and treatment of avian aspergillosis.