Based on the analysis of the cytb gene of mtDNA the taxonomic position was determined of two closely related Bovidae species: American bison (Bison bison) and European bison (Bison bonasus). Reference sequences were determined for both species. In the American bison two variants of cytb gene were identified. Both of them are novel sequences, so far not published in GenBank. For comparison the reference sequences in four another representatives of Bovidae (Polish Red cattle, Zebu cattle, Merino sheep and Polish White Improved goat) were determined. Phylogenetic reconstructions were obtained by the neighbour-joining method (N-J) on all mutations and on changes with removed mutations In third position. Topology of phylogenetic trees showed that American bison and European bison form a separate nodes. The molecular data presented corroborate the results of earlier studies showing that although the two species in question are very closely related, their classification as subspecies is rather questionable. The fact that nodes for American as well as European bison show relatively high bootstrap scores supports this hypothesis.