Bovine theileriosis, caused by T. annulata, results in high morbidity and mortality rates, as well as severe financial losses for the livestock industry in Egypt. In this study, fifty cattle were utilized. Whole blood samples were collected for laboratory analysis. Giemsa-stained blood films were employed to detect Theileria infection. PCR was used to evaluate various target genes, like 30-kDa and Cyto B of T. annulata. Nine (18%) samples tested positive for piroplasm of Theileria by microscopic examination of blood film. Twenty-one (42%) of the analyzed samples tested molecularly positive based on the 30-kDa gene (N516/N517), while 10 (20%) samples were positive based on the Cyto B gene. In our study, we carried out DNA sequencing and phylogenetic analysis of T. annulata using the Cyto B gene. Phylogenetic analysis of the Cyto B gene of the Egyptian strain of T. annulata (Assiut) revealed a nucleotide identity ranging from 96.16% to 98.92% with T. annulata strains from various Egyptian governorates (Sharkia and Qalyubia), as well as from Sudan, Tunisia, Turkey, Iran, and India. The obtained isolates were closely clustered with an isolate from Sudan (accession number LC431533). We identified thirty-point changes at the amino acid sequences. There was substantial variance (P<0.05 and P<0.01) between age and sex of tested cattle, respectively, and percentages of T. annulata infection. The data obtained from our study on the characterization of the Cyto B gene of T. annulata in Assiut Governorate suggest that the Cyto B gene may be used as a genetic marker to identify resistant isolates of T. annulata.