Buparvaquone is now the principle medicinal drug utilized against tropical theileriosis. The current study was conducted to determine the potential association between the resistance against buparvaquone and the occurrence of mutations in the Cytochrome b (Cyto b) gene of Theileria annulata (T. annulata) in cattle under Egyptian conditions. We sequenced the Cyto b gene of T. annulata of 6 blood samples collected from 1 responding and 5 non-responding cattle to buparvaquone treatment. The analysis revealed that two mutations occurred in codons 253 and 262 within the quinol oxidation (Qo2) drug-binding site of the five resistant isolates. Two mutations in the Qo1 site were found together in the buparvaquone sensitive isolate only. Our findings suggest that the failure of buparvaquone therapy could be attributed to the presence of single or double point mutations at the Qo2 site of T. annulata Cyto b gene.