Bovine piroplasmosis caused byTheileria sergenti is a major cause of economic loss in grazing cattle in Japan. Infected calves show chronic anaemia with intraerythrocytic piroplasms and occasionally die in severe cases. We found that parasite stocks and isolates consist of genetically and antigenically mixed populations. To differentiate parasite populations bearing 3 allelic forms of p32/34, an immunodominant piroplasm surface protein, 3 sets of oligonucleotide primers were designed to amplify either of 3 alleles by polymerase chain reaction (PCR). By using this allele-specific PCR, we found that the majority ofT. sergenti-infected calves in Japan harbored mixed parasite populations bearing C and I type parasites. To controlTheileria infection, we produced 2 vaccine candidates: recombinant baculovirus p32 and synthetic peptide containing Lys-Glu-Lys (KEK) motif. Immunization with either recombinant p32 or synthetic peptide containing KEK sequences with Freund’s complete adjuvant resulted in low parasitemia and reduced the clinical symptoms compared to control calves. Interestingly, the parasite with the p32 allelic form corresponding to the one used as the immunogen was suppressed.