Theileriosis is one of the most economically important tick-borne diseases of cattle and buffaloes in tropical countries and Mediterranean region. The present study investigates 120 cows and 80 buffaloes in Cairo and Giza governorates, Egypt to evaluate PCR assay as sensitive and specific technique and microscopic examination for diagnosis of Theileria sp. Only 5 cows and 2 buffaloes showed clinical signs varied from rise of body temperature, enlargement of superficial lymph nodes, nasal and ocular discharge and sometimes associated with corneal opacity. The infection rate of Theileria in cows and buffaloes was 6 (12%) and 3 (9.3%) in Cairo and 10 (14.2%) and 6 (12.5%) in Giza respectively. The PCR result of examined blood samples of 12 cows and 8 buffaloes were positive for Theileria species by using 989a/990a and Tbs-S/Tbs-A primers where give 1098 pb and 430 pb respectively. None of the samples were positive for Theileria annulata by using N516/N517 primers. All positive samples for Theileria species by using Tbs-S/Tbs-A primers were used as a template and amplified by using Ta-S/Tbs-A primers specific for Theileria annulata, 10 out of 12 and 5 out of 8 blood samples of cow and buffaloes respectively were positive for Theileria annulata and give 193 bp.