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Seabird rehabilitation is a valuable strategy to mitigate the impacts of oil pollution and other anthropogenic factors, and can significantly contribute to the conservation of penguins. However, infectious diseases such as avian malaria (Plasmodium spp.) can hamper the success of rehabilitation efforts. We combined morphological and molecular diagnostic methods to investigate the epidemiology and pathology of Plasmodium in Magellanic penguins (Spheniscus magellanicus) at rehabilitation centers along 2500 km of the coastline of Brazil. True prevalence of malarial parasites was estimated between 6.6% and 13.5%. We identified five species, three of which had not been described infecting penguins (P. cathemerium, P. nucleophilum, P. unalis); an additional five distinct Plasmodium lineages were also distinguished, and albeit unidentified these clearly correspond to species that also have not yet been reported in penguins. Our results indicate that the diversity of plasmodia that may infect these birds is greater than previously recognised. Considering the well-defined seasonality observed in this study, it is clear that rehabilitation centers could benefit by narrowing their preventative efforts on penguins maintained or admitted during the Austral spring-summer, particularly by preventing mosquitoes from coming into contact with penguins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0160-9) contains supplementary material, which is available to authorized users.