Background: Theileriosis is a notable tick-borne illness commonly found in tropical and subtropical regions, leading to significant annual economic losses. This research aimed to investigate the problem of drug resistance in dairy cattle suffering from theileriosis. Methods: In the spring and summer of 1403, 90 blood samples were gathered from dairy cattle that showed inadequate responses to buparvaquone treatment in Roshtkhar area. Initially, blood smears stained were prepared and examined under a microscope, followed by the extraction of DNA from EDTA blood samples, which were subsequently analyzed using specific PCR techniques for Theileria.The PCR-positive samples underwent testing with specific primers that target the cytochrome B gene of Theileria annulata, and subsequently, ten blood samples that exhibited a good positive reaction were submitted for DNA sequencing. The sequence analysis was conducted on PCR products based on the amplification of the cytochrome B gene to identify gene mutations in the isolated samples. Results: Microscopic and PCR examinations of the blood samples indicated the presence of Theileria infection in 73 and 75 samples, respectively. Sequencing analyses revealed a total of thirteen gene mutations in the nucleotide sequences of four samples when compared to the references strain. Among these mutations, eight were found within the Q01 region, while the remaining mutations were located outside the Q01 and Q02 regions. The highest number of gene mutations was observed in samples No. 2 and No. 4. In this study, only sample No. 2 contained seven newly formed mutations, with six of these mutations leading to changes in amino acids. Conclusion: Three gene mutations in this sample were identified within the Q01 region, which may indicate a resistance of T. annulata to the Butalex drug. Considering the widespread occurrence of this disease in Iran, it is recommended to implement management strategies for treatment and control of this disease within the country.

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