Here we describe the in vitro isolation, propagation, and characterization of a Theileria species from roan antelope (Hippotragus equinus). Cultures were initiated using parts of a prescapular lymph node of an infected roan antelope. After 16 days of culture propagation, the first subculture was carried out; thereafter, subcultures were carried out twice a week. Standard methods for the cultivation of Theileria macroschizonts were applied. DNA was extracted from culture material and a partial polymerase chain reaction amplification of the 18S ribosomal RNA (rRNA) gene was carried out using Theileria genus-specific primers. It has been shown that Theileria sp. (roan) had high levels of nucleic acid identity with sequence data of the 18S rRNA gene of a Theileria sp. previously isolated from a sable antelope. The phylogenetic analysis showed that this isolate is closely related to several undescribed Theileria spp. which have previously been identified from a dog and some other antelope species in South Africa.