Two fragments of mitochondrial DNA (mtDNA) of the cytochrome b gene (137 bp and 167 bp) were successfully isolated and sequenced from antlers and bones of five specimens of the Giant Deer (Megaloceros giganteus) to examine the phylogenetic position of Megaloceros giganteus within the family Cervidae. This is the first report on ancient DNA (aDNA) sequences from Megaloceros giganteus. A phylogenetic analysis based on parameter-rich models describes the evolutionary relationships between five individuals of fossil Megaloceros giganteus and 37 individuals of 11 extant species of the family Cervidae. The results support a “Cervus–Megaloceros” clade. The phylogenetic positions of sympatric Megaloceros and Cervus elaphus specimens in particular indicate either that the Megaloceros mtDNA gene pool did not evolve for a substantial time period as an entity distinct from Cervus elaphus until its extinction, or that Megaloceros contributed mtDNA to Cervus elaphus or vice versa. The results of this study allow the conclusion that the European Megaloceros giganteus is more related to its modern regional counterparts of the species of Cervus elaphus than recent claims have suggested.