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Emerging infectious diseases in wildlife are increasingly associated with animal mortality and species declines, but their source and genetic characterization often remains elusive. Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis ( Bd ), has been associated with catastrophic and well-documented amphibian population declines and extinctions at the global scale. We used histology and whole-genome sequencing to describe the lesions caused by, and the genetic variability of, two Bd isolates obtained from a mass mortality event in a captive population of the threatened Chilean giant frog ( Calyptocephalella gayi ). This was the first time an association between Bd and high mortality had been detected in this charismatic and declining frog species. Pathological examinations revealed that 30 dead metamorphosed frogs presented agnathia or brachygnathia, a condition that is reported for the first time in association with chytridiomycosis. Phylogenomic analyses revealed that Bd isolates (PA1 and PA2) from captive C. gayi group with other Bd isolates (AVS2, AVS4, and AVS7) forming a single highly supported Chilean Bd clade within the global panzootic lineage of Bd ( Bd GPL). These findings are important to inform the strengthening of biosecurity measures to prevent the impacts of chytridiomycosis in captive breeding programs elsewhere., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alvarado-Rybak, Acuña, Peñafiel-Ricaurte, Sewell, O'Hanlon, Fisher, Valenzuela-Sánchez, Cunningham and Azat.)