Background <p><i>Theileria annulata</i>, a tick-borne protozoan that causes tropical theileriosis, poses a serious threat to livestock production in endemic regions. The emergence of resistance to buparvaquone, the primary chemotherapeutic treatment, has been attributed to acquired mutations in the cytochrome b (<i>Cytb</i>) gene, with identical resistance-associated polymorphisms observed in both laboratory-adapted strains and field isolates from China.</p> Methods <p>A dual probe-specific real-time polymerase chain reaction (PCR) assay was developed to detect point mutations in the <i>Cytb</i> gene. The specificity, sensitivity, and reproducibility of the assay were validated, and its field applicability was evaluated via cattle blood samples (<i>n</i> = 531) collected from five endemic Chinese provinces.</p> Results <p>Six point mutations were identified in the <i>Cytb</i> gene, and the developed dual probe-specific real-time PCR assay simultaneously detected <i>T. annulata</i> infection and distinguished between the buparvaquone-sensitive and buparvaquone-resistant genotypes. The assay demonstrated a detection limit of 1 × 10¹ copies/μl, high specificity, and satisfactory repeatability, with results consistent with those of Sanger sequencing. Field screening revealed a 21.7% (115/531) prevalence of <i>T. annulata</i> and a 4.3% (23/531) occurrence of resistant genotypes. Moreover, two-dimensional scatterplot visualization enabled clear genotype discrimination without post-PCR processing.</p> Conclusions <p>The developed dual probe-specific real-time PCR assay enables efficient detection of buparvaquone-resistant genotypes, providing important implications for guiding the treatment of tropical theileriosis and enhancing epidemiological surveillance of emerging resistance in endemic regions.</p> Graphic abstract <p></p>