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Aim: To investigate the cause of a high seroprevalence of antibodies to Plasmodium spp known to cause avian malaria, but extremely low levels of observed infection, in yellow-eyed penguins, Megadyptes antipodes., Methods: A polymerase chain reaction (PCR) test specific for malarial parasites was applied to DNA extracted from blood samples collected from 143 yellow-eyed penguins from an area where seroprevalence for malarial antibodies was known to be high but no parasites were observed in blood smears., Results: None of the samples tested positive for malarial parasite DNA using the PCR test. Assuming a sensitivity of 90% for this test, this means that prevalence of infection was 95% likely to be <2.3% in this population during this sampling period., Conclusions: Serological studies of a population of adult yellow-eyed penguins indicated a high level of exposure to avian malaria parasites, but a correspondingly high level of infection was not observed and no evidence of malarial parasite DNA was found in the current study. Discrepancies between these findings and historical records of Plasmodium spp found in blood smears and post mortem may be explained either by inaccuracy of the serological test used, or by infection occurring in juveniles which is subsequently cleared in surviving adults.