Penguins represent unique morphology and inhabit widely in the Antarctic and the Sub-Antarctic Islands. They have adapted for ecologically diverse environments from Antarctic zone to subtropical area. There are still some debates about the classification of penguin species. We previously reported that polymorphisms in the MHC class II genes were useful in tracing phylogenetic relations among penguin species. However, there is no report on the diversities in the MHC class I gene. In this study, we determined nucleotide sequences spanning from intron 2 to exon 4 of MHC class I gene from 29 penguins belonging to 7 different species and obtained a total of 34 alleles. Phylogenetic analysis revealed that alleles from 4 penguin species placed in the genus Spheniscus, Spheniscus humboldti, S. magellanicus, S. demersus and S. mendiculus, were not separated but clustered into one, whereas alleles from three other species, Pygoscelis papus, Aptenodytes forsteri and Megadyptes antipodes, separately clustered depending on the species. These observations suggested that MHC class I diversities are useful genetic markers to investigate the phylogenetic relations of penguin species.