Following a study on molecular epizootiology of Hepatozoon canis and piroplasmids (Babesia spp. and Theileria spp.) in southern Europe, newly obtained sequences of 18s rRNA gene were used for phylogenetic analysis. Partial sequences were analysed in isolates showing high degree of homology (>99%) with previous GenBank entries: H. canis, B. canis vogeli, B. equi (two isolates, Spain1 and Spain2), T. annulata and Theileria sp. The complete gene sequences were used for B. ovis and B. bovis, that showed lower homology (<95%) with rapport to previously reported species or isolates. A first set of phylogenetic trees constructed with partial 18s rRNA sequences showed that most European isolates clustered unambiguously with previously described species, so that minor sequence dissimilarities found are due probably to strain variations. The second set of phylogenetic trees was made using the complete 18s rRNA sequences of 44 species from GenBank and the newly sequenced B. ovis and B. bovis. The analysis revealed for the first time a division of piroplasmids in five clades: (1) B. microti group, with B. rodhaini, B. felis, B. leo, B. microti and T. annae (proposed name for the group, without taxonomic value: Archaeopiroplasmids), (2) Western USA Theilerid-like group (proposed name: Prototheilerids), (3) Theileria group, containing all Theileria species from Bovinae (proposed name: Theilerids), (4) A first group of Babesia species including B. canis and B. gibsoni from canids together with B. divergens and B. odocoilei (proposed name: Babesids), (5) A second group composed mainly by Babesia species from ungulates: B. caballi, B. bigemina, B. ovis, B. bovis and Babesia sp. from cow (proposed name: Ungulibabesids). The bootstrap support obtained with several analytical procedures for this new dicotomy of Babesiidae was always very high. Taking into account the present phylogenetic analysis and additional paleogeographic, parasitological and zoological evidences, two hypothesis on the origin and evolution of piroplasmids groups are presented.