The Gentoo Penguins Pygoscelis papua has been classified into two subspecies, Northern P. papua papua and Southern P. papua ellsworthi. In Japan, where a breeding program exists for Gentoo Penguins, the Japanese Association of Zoos and Aquariums (JAZA) has recognized three categories of species in its pedigree ledger: Northern (from the islands of South Georgia), Southern (from the South Shetland Islands), and Unknown (when their origins are not clear). Phylogenetic analyses of the mitochondrial DNA (mtDNA) D-loop region of Gentoo Penguins in their natural habitat have differentiated them as belonging to the Kerguelen Islands (Indian Ocean), Falkland Islands (Atlantic Ocean), and South Georgia (Atlantic Ocean), and categorized some as Northern and others as Southern subspecies. However, the identification of a third subspecies has been suggested. Therefore, reclassification of captive Gentoo Penguins using phylogenetic analysis is necessary for domestic subspecies pedigree management. In this study, we determined five mtDNA haplotype sequences using seven Gentoo Penguins (considered morphologically as three Northern, three Southern, and one Unknown) and characterized their phylogenetic relationships among subspecies and among geographical distributions including 56 published mtDNA sequences. The phylogenetic tree showed three major lineages, Kerguelen Islands, Falkland Islands, and Scotia Arc/Antartica. The five haplotype sequences of the seven captive Gentoo Penguins were classified unexpectedly to one lineage, the Scotia Arc/Antartica, which contains the nucleotide sequences derived from Southern P. papua ellsworthi. Therefore, the mtDNA phylogeny of the seven captive penguins suggests that all captive Gentoo Penguins in Japan belong to the Southern subspecies. If so, then more comprehensive genetic testing of Gentoo Penguins in Japanese aquariums and zoos is required for the management of successful breeding programs.