Background: Anaplasma marginale ssp. centrale (A. centrale) exhibits low pathogenicity and therefore is used as a live vaccine against bovine anaplasmosis in several countries. During production of the vaccine, accidental contamination with Anaplasma marginale (A. marginale) is a risk that can jeopardize the entire batch of vaccine. Due to limitation of microscopic examination to detect low levels of parasitaemia, the present study aims to standardize a polymerase chain reaction assay using primers for the msp4 gene of Anaplasma sp. for detecting and differentiating with greater sensitivity and specificity the species of A. centrale and A. marginale in blood samples from experimentally infected cattle. Materials, Methods & Results: The DNA extraction was performed from frozen blood. Erythrocytes infected with known A. centrale, A. marginale served as positive control and the erytrocytes infected with Babesia bovis and Babesia bigemina served as the negative control polymerase chain reaction. PCR was standardized from annealing temperature variations of the primers, magnesium chloride concentration, amounts concentration of primers and DNA concentration of rickettsiae. By PCR method, it was analyzed the DNA from blood samples of 13 cattle positive to A. marginale by microscopic examination from smear stained with Giemsa. The PCR assay was specific for A. centrale and A. marginale, presented 100% identity without presenting cross-reactivity with other bovine hemoparasites. The detection limits of the PCR were 0.25 pg and 0.125 pg of DNA for detection of A. centrale and A. marginale DNA respectively. Discussion: A. marginale is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia being considered the main agent of bovine anaplasmosis.The strain of A. centrale by having a low pathogenicity has been used since the years 1900 a live vaccine with the aim of decreasing the clinical signs associated with exposure to field strains of A. marginale in some countries. The vaccine production involves exposure of calf via inoculation of erythrocytes infected with the A. centrale strain, which need to be free from A. marginale and other bovine hemoparasites. Because the vaccine A. centrale be produced in splenectomized calves, a rigorous care as accidental contamination mainly by blood-sucking insects should be performed. Since a contamination during the isolation of live A. centrale vaccine can compromise the entire batch of vaccine. There are some limitations to the current methods used for the detection of a possible contamination of live vaccine A. centrale. The serological tests do not allow differentiation between Anaplasma species, because of the similarity of their antigens and cross-reactions with other hemoparasites can take place. Microscopic examination of blood differentiates A. centrale from the A. marginale strain by of the characteristics and morphological distribution of the parasite in erythrocytes. However, this procedure does not detect pre-symptomatic and carrier animals, since its sensitivity is in the range of 106 infected erythrocytes per mL of blood. In contrast, the molecular biology have potential to detect low levels of blood DNA and differentiate A. centrale from A. marginale through the presence of major surface protein 4 (MSP4), and thus allows the verification of the purity of A. centrale vaccine.