To investigate the presence of T. annulata and T. buffeli/orientalis complex in the blood of 172 cattle selected from 9 different regions of Kırşehir.Genes for the merozoite surface antigen (Tams 1) and the major piroplasm surface protein (MPSP) were amplified with multiplex- PCR for T. annulata and T. buffeli/orientalis, respectively.By multiplex-PCR examination 4 (2.32%) samples were positive for T. annulata whereas none of the samples were positive for T. buffeli/orientalis complex.In this study, it is concluded that simultaneous diagnosis of Theileria species by using multiplex-PCR is more practical than the investigation of species individually by using classical PCR. We also believe that a more accurate epidemiological data is achieved with the use of moleculer diagnosis methods such as multiplex-PCR, which eliminates the disadvantages of the traditional methods used in the diagnosis of parasitic diseases.