The aim of this study was to diagnose Theileria annulata infected cattle by microscopy, IFAT and nested PCR and to compare the sensitivity and specificity of these diagnostic methods. A total of 147 blood and sera samples were collected from healthy cattle in four localities of Polatlı district, Ankara, where tropical theileriosis is prevalent. Examination results revealed positivity rates of 31.3%, 44.9% and 61.2% for microscopy, IFAT and nested PCR, respectively. It was shown that nested PCR is more sensitive and specific when compared to microscopical examination and serological findings. It was also shown that nested PCR is more sensitive than single step PCR. This study, which is the first work utilising nested PCR in the detection of T. annulata in Turkey, showed that nested PCR is more sensitive and specific when compared to microscopy and IFAT and can be used in further epidemiological studies