In order to develop a method for detecting and identification of Theileria annulata, the specific primers from the major merozoite-piroplasm surface antigen sequence of Theileria (Tams1) were used to detect the T. annulata by nested-PCR technique. The assay provides a valuable tool for the identification of Theileria annulata directly from clinical samples and enables determination of the infecting species by a facile technique with high sensitivity and specificity power. The sensitivity of the PCR was determined up to 1.34 pg infected DNA, and specificity of the PCR was confirmed by DNA sequencing. The Tams1 nested-PCR assay will facilitate parasite infection follow up and might improve diagnosis and therapeutic approach of bovine tropical theileriosis. Moreover, multiple alignment and phylogenetic analysis of Tams1 sequences of available strains/isolates showed that there is a particular restriction site in T. annulata Iran-vaccine strain could be recognized by AvaII enzyme. These findings can be used in further disease control and prevention program as well as epidemiological studies.