In 1911, Sir Arnold Theiler isolated and described a parasite that was very similar to A. marginale but which was more centrally located within the erythrocytes of the host cells, and was much less pathogenic than A. marginale. He named the parasite A. marginale variety centrale. The name A. centrale, referring to the same organism, was published in Validation List no. 15 in 1984, but the publication was based on an erroneous assumption that Theiler had indicated that it was a separate species. Many authors have subsequently accepted this organism as a separate species, but evidence to indicate that it is a distinct species has never been presented. The near full length 16S rRNA gene sequence, and the deduced amino acid sequences for groEL and msp4 from several isolates of A. marginale and A. centrale from around South Africa were compared with those of the A. marginale type strain, St Maries, and the A. centrale Israel strain and other reference sequences. Phylogenetic analyses of these sequences demonstrated that A. centrale consistently forms a separate clade from A. marginale, supported by high bootstrap values (≥90%), revealing that there is divergence between these two organisms. In addition, we discuss distinctive characteristics which have been published recently, such as differences in Msp1a/Msp1aS gene structure, as well as genome architecture that provide further evidence to suggest that A. centrale is, in fact, a separate species. Our results, therefore, provide evidence to support the existing nomenclature, and confirm that A. centrale (ex Theiler 1911) (Ristic and Kreier, 1984) is, indeed, a distinct species.