One hundred-fifty Yellow-eyed penguins (Megadyptes antipodes) of 8 populations, 20 Little blue penguins (Eudyptula minor) of 2 populations, 9 Rockhopper penguins (Eudyptes chrysocome), and 5 Adelie penguins (Pygoscelis adeliae) from New Zealand (NZ) and Antarctica (latitude range; 39 degrees 30' S - 77 degrees 13' S) were tested by indirect enzyme-linked immunosorbent assays (ELISA) for fungal Aspergillus spp. IgG. One hundred-ten of 184 (60%) penguins were seropositive, and seroprevalence was related to the latitudinal location of a population. All E. chrysocome from Campbell Island and Antarctica P. adeliae were seronegative. The seroprevalence varied significantly (p < 0.01) from 14% to 80% among M. antipodes populations, with a mean of 60%. Seroprevalence was 100% for E. minor populations. The mean absorbances varied significantly (p < 0.01) from 0.32 to 0.70 for M. antipodes populations, and were 0.44 and 0.51 for E. minor. Seventeen penguins (11 E. minor, 6 M. antipodes) of 110 (15%) seropositive birds had absorbance within the 95.0% absorbance confidence interval of fatal aspergillosis of captive Jackass penguins (Spheniscus demersus).