Simple SummaryAspergillosis is a fungal respiratory infectious disease in humans and animals and a major cause of mortality in captive penguins. The causative agents of this disease are members ofAspergillusspecies. These fungi are commonly found in the environment. Therefore, rearing conditions and countermeasures in facilities are important to preventing aspergillosis. However, their efficacy has not been investigated sufficiently. In addition, a nationwide survey on the occurrence of aspergillosis, rearing conditions, and countermeasures has not been performed for several decades in Japan. Therefore, this study evaluated the incidence of and preventive measures against aspergillosis in Japanese zoos and aquariums that rear penguins. The incidence of aspergillosis was significantly high in facilities where penguins were housed outdoors, in contact with soil, or moved outside of the rearing enclosure. Overall, 76% of dead penguins had been at individual risk and 54% had been kept in uncomfortable environments. Aspergillosis was thought to occur when individual risk factors and uncomfortable environmental factors were present along with the risk ofAspergillusexposure. Recognizing these risks and implementing appropriate measures to prevent contact withAspergillus-contaminated soil are necessary to further reduce aspergillosis-related mortality in penguins.AbstractWe surveyed the facilities that were members of the Japan Association of Zoos and Aquariums to clarify the incidence of aspergillosis, which is a major cause of death in captive penguins, and to discern effective preventive measures. Responses were obtained for 2910 penguins in 64 facilities; 73 penguins (2.5%) in 35 facilities had died from aspergillosis during the past 5 years from April 2016 to March 2021. Answers to questions about the rearing environment indicated that aspergillosis occurred significantly more often in facilities where penguins were reared outdoors, were in contact with soil, or were moved outside of the rearing enclosure. Answers to questions about their dead penguins indicated that 76% may have been at individual risk (e.g., young age, old age, molting period, and breeding season) and 54% were thought to be reared in uncomfortable environments (e.g., high temperature, high humidity). Aspergillosis may occur when individual risk factors and uncomfortable environmental factors are added to the risk factors of exposure toAspergillus, such as the presence of soil. These conditions must be recognized as risk factors for aspergillosis, and appropriate preventive measures, such as avoiding penguin contact with the soil whereAspergillusis expected to be present, can minimize aspergillosis-related deaths.