theileria equi is a tick-borne hemoprotozoan parasite and one of the causative agents of equine piroplasmosis. Data on T. equi infection in donkeys are scarce in Assiut governorate, Egypt. This study was conducted on the prevalence of T. equi infection in donkeys by using micropscopic examination and polymerase chain reaction (PCR) assay. Out of 50 donkeys, blood samples examined for T. equi, 14% (7/50) were positive by microscopic examination and 38% (19/50) by PCR. Prevalence of T. equi had high significant differences between microscopic examination and PCR assay. Prevalence of T. equi in male donkeys was 13.6% (3/22) by microscopic examination and 36.4% (8/22) by PCR. In female donkeys it was 14.3% (4/28) by microscopic examination and 39.3% (11/28) by PCR. No significant difference between the prevalence rate of infection in males and females were recorded. Microscopic examination of donkeys' blood smears stained with Giemsa stain revealed forms of theilerial schizogony in lymphocytes (Kochs blue bodies) and intra-erythrocytic relatively small, less than 2 μm long pear-shaped and ring shaped merozoites of T. equi. The B1 gene specified for T. equi was detected by PCR in 19 blood samples. All positive samples with microscopic examination were also positive with PCR. The sensitivity, specificity and accuracy of PCR with respect to blood film examination were 61.3%, 100% and 82%, respectively. So, PCR was found to be more sensitive, specific and accurate than blood film examination.The present results suggest that T. equi parasite is widely spread in Assiut governorate due to high exposure to ticks.