Theileria species are tick-borne protozoal parasites that infect many domestic and wild animals worldwide. Theileria annulata (T. annulata) is the most economically important species affecting cattle and buffaloes. This work was aimed to detect the prevalence of Theileria species in cattle and buffaloes during the period extended from January 2018 till December 2018 at different localities of Sharkia Governorate, Egypt. Blood samples were collected randomly from 174 (86 cattle and 88 buffaloes) apparently healthy and clinically infected animals. Samples were examined microscopically using Giemsa stained blood films. The prevalence of T. annulata was 34.88% and 32.95% in cattle and buffaloes respectively. T. annulata infection was higher in male (44.68% &48.08%) than female (23.08% &11.11%) in cattle and buffaloes respectively. Furthermore, T. annulata prevalenceinfection was higher in old animal (>6 years) (61.29% &48.48%) than 1-6 years (25% &37.5%), 1- 2 months (18.18% & 20%) and 3-12 months (16.67% &21.62%) in cattle and buffaloes respectively. The tick infestation status showed a potential risk factor in developing infection. The PCR product length was 721 bp from T. annulata positive samples using the specific primer. Sequencing of the PCR products and subsequent blast analysis to detect their identities were clarified with the previous studies.