BACKGROUND: Ovine theileriosis is an important hemoprotozoal disease of sheep and goats in tropical and subtropical regions which causes high economic loss in the livestock industry. OBJECTIVES: The aim of this study was the differential detection of Theileria species in sheep using PCR method. METHODS: Two hundred blood samples of sheep were investigated in order to differentially diagnose Theileria species. DNA was extracted from blood samples and DNA samples were amplified using specific primers designed for 18S rRNA, TamS1 and TaSp genes. RESULTS: In this study, from 200 examined samples, 42 samples (21%) were infected by Theileria spp. and none of them were infected by Babesia spp. Moreover, from these 42 positive samples, 24 samples (57.1%) were only infected by T. ovis. 12 samples (28.5%) were only infected by T. lestoquardi, 2 samples (4.7%) were only infected by T. annulata and 4 samples (9.5%) were simultaneously infected by T. lestoquardi and T. ovis. The results of nucleotide sequencing showed that PCR product of 18S rRNA from T. lestoquardi has 99 and 95% similarity with T. annulata and T. ovis respectively. T. lestoquardi and T. annulata showed 86% similarity. Also TaSp gene of T. ovis in comparison with T. annulata and T. lestoquardi showed 96 and 86% similarity, respectively. CONCLUSIONS: In the present study could be shown that the two genes (TamS1 and TaSp) from examined three genes could be used for Theileria species specific diagnosis by PCR.