The aim of this study was to detect and differentiate Theileria annulata and T. lestoquardi (hirci) by PCR. Members of the genus Theileria are tick-borne hemoprotozoan parasites those cause fatal and enervating diseases of cattle and sheep in Iran . In order to develop a specific method for detecting and identification of Theileria species, specific primers from the surface protein (SP) sequence were designed that allowed the specific diagnosis of T. annulata and T. lestoquardi infection simultaneously. Theileria surface protein genes have consensus and variable sequences regions that allowed us to design the common primers for both species, which amplified two different PCR products. The results of this study demonstrated that a novel, simple, and high specific PCR for detecting and identifying T. annulata and T. lestoquardi infection.