Fifty-eight frozen postmortem lung and air sac samples were collected from penguins housed at 21 zoological collections throughout the United Kingdom, from 2007 to 2018. Aspergillus fumigatus, a significant respiratory pathogen of penguins in captivity, was isolated from 15 of the 22 penguins with gross lesions. Of the penguins with gross lesions of aspergillosis at postmortem examination, the pathogen was cultured from 63.6% (15 of 22) of frozen samples. Aspergillus fumigatus was cultured from 2.7% (1 of 36) of tissues collected from penguins without gross lesions at postmortem. Additionally, of 18 fresh samples that cultured A. fumigatus at the time of postmortem, 15 samples (83%) yielded isolates that were successfully cultured from frozen tissue. Minimum inhibitory concentration (MIC) data demonstrated that all isolates were susceptible to terbinafine and voriconazole, and all were resistant to itraconazole, using published MIC cutoff values. Comparison isolates from fresh tissues had identical antimicrobial susceptibility to isolates from the same tissues after being frozen. This study demonstrates that A. fumigatus can be isolated from frozen respiratory tissues of penguins, even after freezing for more than 10 yr.