Tick-borne bovine anaplasmosis, caused by the obligate intracellular pathogen Anaplasma marginale(Rickettsiales: Anaplasmataceae), is a major constraint to cattle production in tropical and subtropical regions. From Madagascar, clinical cases were published but data based on molecular methods regarding the prevalence and genetic diversity of this pathogen on the island are lacking. The aims of this study were to investigate (1) the prevalence of A. marginalein Malagasy zebu cattle (Bos indicus)and their ticks with a species-specific real-time PCR, (2) the genetic diversity of A. marginalebased on tandem repeats and microsatellites of the msp1α gene, and (3) the phylogenetic relationship between A. marginaleisolates from Madagascar and strains found worldwide. Two hundred fourteen blood samples and 1822 ticks from 214 zebu cattle were collected. Rhipicephalus (R) microplus(40.2%) and Amblyomma (A) variegatum(59.8%) were identified on the cattle. A. marginaleDNA was found in 89.7% of the examined zebu cattle and in 62.3% of the examined ticks.