This study aimed to develop a multiplex PCR for simultaneous detection of Theileria annulata, Babesia. bigemina and Anaplasma marginale infections and their carrier status in cattle reared in the different agro-geoclimatic regions of Tamil Nadu, India. The primers for this mPCR were designed from the Cytochrome b gene, 18s rRNA and 16s rRNA for T.annulata, B.bigemina and A.marginale respectively. The specificity of this PCR was 100 per cent for detecting T. annulata and A. marginale and 96 per cent for B.bigemina. The sensitivity of this PCR for detecting the DNA (ng/ml) of T.annulata, B.bigemina and A.marginale was 1.10, 2.09 and 2.59, respectively. Investigations with field samples revealed successful detection of single, double and multiple infections of T.annulata, B.bigemina and A.marginale.