Anaplasma marginale is a rickettsial pathogen responsible for bovine anaplosmosis, the acute disease in cattle herds which is associated with anemia, fever, rapid loss of milk production and weight, abortion, and, in some cases, death of the infected cattle. Anaplasma marginale is transmitted by ticks and biting insects. Diagnosis of bovine anaplasmosis is made by microscopic examination of blood smears stained with Giemsa stain, but this method is not useful to detect presymptomatic animals. Several serological tests have used extensively for epidemiological studies, but they do not discriminate between different Anaplasma species. A real-time polymerase chain reaction (PCR) combines high specificity with accurate measurement of DNA copy number and allows quantification of the targeted pathogen DNA. The goal of this study was to develop a real-time PCR assay for differential detection of A. marginale in the blood of cattle.