BACKGROUND: Theileriosis is a blood protozoan disease with high mortality in cattle in tropical and subtropical regions of the world. Several studies were conducted to identify Theileria species in infected cattles and vector ticks by microscopic examination. However, microscopic technique has lower sensitivity compared to molecular method.                  OBJECTIVES: This study was carried out to identify Theileria species and its carriers in cattles of Yazd city, sing semi nested PCR. METHODS: Between June to September of 2012, 100 EDTA blood samples and 249 ticks were collected from Holstein breed with no history of vaccination against the ileriosis in Yazd area. The collected samples were transported to the laboratory, then prepared the blood smears and stained with Giemsa method. Also, the collected ticks were separated into 50 tick pools, according to their species. Then their salivary glands were removed using stereomicroscope in 0.85% saline. DNA of blood and salivary glands was extracted using a commercial kit and analyzed by Semi-nested PCR. PCR-RFLP was also used to differentiate Theileria lestoquardi from Theileria annulata in positive samples of ticks. RESULTS: Ring forms of Theileria spp. were found in 4 (4%) of blood smears. All ticks were Hyalomma a.anatolicum. Results of PCR were indicated that 11 (11%) of blood samples and three pools of tick’s salivary glands were infected with Theileria. Theileria annulata were only detected in all positive samples by Semi-nested PCR. The results of PCR-RFLP using MspI enzyme showed that the tick’s salivary glands were negative to Theileria lestoquardi infection. CONCLUSIONS: Based on the results, it is concluded that Theileria annulata and Hyalomma a.anatolicum are important agent and vector tick of tropical theileriosis in dairy cattles of Yazd area.