Tropical theileriosis is a progressive bovine lymphoproliferative disease caused by the intracellular protozoan parasite Theileria annulata. In this study 138 blood samples and 289 ticks were collected and examined from cattle that belonged to 10 randomly selected flocks. The Tbs-S/Tbs-A primer set was used for PCR amplification of Theileria spp. and the Ta-S/Tbs-A specific primer set was used in semi-nested PCR technique for detection of T. annulata. Blood smears of each case were examined by Giemsa staining method. The semi-nested PCR accurately revealed 22 (15.94 %) positive samples; whereas Giemsa staining method could detect 15 (10.86 %) out of 138 blood samples. The examination of 289 ticks by semi-nested PCR revealed that, 32.86 % of Hyalomma anatulicum anatulicum, 26.47 % of Hyalomma anatulicum excavatum and 22.42 % of Hyalomma asiaticum asiaticum, were infected with T. annulata. The results suggest that H. anatulicum anatolicum may play a major role in transmission of T. annulata infection in Iran. The results indicated that the Giemsa staining method, having low sensitivity, while the semi-nested PCR technique can be used as a gold standard method for this purpose.