Bovine anaplasmosis caused by the intraerythrocytic rickettsia Anaplasma marginale is the most prevalent tick-borne disease of cattle worldwide. The most efficient method to control anaplasmosis is by vaccination using live Anaplasma centrale, a closely related species or subspecies of low pathogenicity that is capable of inducing significant protection against the more virulent A. marginale. In the present study, we applied PCR assays to detect and discriminate field infection with A. marginale from A. centrale persistently infected vaccinates. Direct and one-stage nested PCR were based on A. centrale mbp58 specific sequence, with the assay sensitivity level of 0.00001% for nested PCR performed in a single amplification step. Size polymorphism in the A. marginale msp1 alpha gene among strains was used to design a PCR capable of discriminating between the Israel T and NT strains of A. marginale and the encoded MSP1a size polymorphism was confirmed by immunoprecipitation. The detection of A. centrale in 72% of vaccinated field-grazing cattle clearly indicated that the majority of vaccinated cattle remain carriers. A. marginalewas detected in 64% of these vaccinated cattle, demonstrating that, as expected, natural transmission occurs within the endemic region. The lack of severe A. marginaleoutbreaks in this region, despite ongoing transmission, is consistent with protection being provided by widespread vaccination with A. centrale.