Echinococcus felidis had been described in 1937 from African lions, but was later included in Echinococcus granulosus as a subspecies or a strain. In the absence of any genetic characterization, most previous records of this taxon from a variety of large African mammals remained unconfirmed due to the lack of diagnostic criteria and the possible confusion with the sympatric E. granulosus sensu stricto, Echinococcus ortleppi and Echinococcus canadensis. In this study, we obtained taeniid eggs from lion feces in Uganda and amplified DNA from individual eggs. Mitochondrial and nuclear DNA sequences showed similarities with those of other Echinococcus spp., but high values of percentage divergence of mitochondrial genes indicated the presence of a distinct species. In a second step, we compared this material with the preserved specimens of adult E. granulosus felidis, which had been identified morphologically approximately 40 years ago in South Africa. All DNA fragments (<200 bp) that could be amplified from the adults showed 100% similarity with the Ugandan material. In the phylogenetic tree of Echinococcus which was constructed from the mitochondrial genes, E. felidis is positioned as a sister taxon of E. granulosus sensu stricto. The data obtained will facilitate the development of diagnostic tools necessary to study the epidemiology of this enigmatic parasite.