Tropical theileriosis is considered one of the most economically important tick borne diseases that cause significant mortality and morbidity to livestock. In the context of epidemiological studies on livestock in Egypt, this report investigated the spread of Theileria annulata among diseased farm cows (Bos indicus) over one year. Blood samples collected from 130 cows were investigated by routine staining and 64 samples were investigated by PCR assay using two different probes targeting tams-1 gene. Microscopy revealed the infection of 33.8% of animals with Theileria while PCR detected infection in 51% of animals with one primer pair and the other primer pair detected the infection in 31% of animals. Combined PCR results indicated the infection of 68.8% of animals with T. annulata. Seasonal fluctuation of parasite infection was evident with the highest infection percentage and prevalence recorded during summer based on both microscopy and PCR data. For the first time, the current study reports the presence of two T. annulata isolates based on tams-1 gene partial sequence in Egypt. Targeting polymorphic genes for parasite detection may result in underestimation of infection and target gene diversity has to be considered. The high infection with these pathogens in the clinically ill cows necessitates implementing serious programs to minimize their economic burden on the Egyptian farming industry.