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. marginale in Malagasy zebu cattle (Bos indicus) and their ticks with a species-specific real-time PCR, (2) the genetic diversity of A. marginale based on tandem repeats and microsatellites of the msp1α gene, and (3) the phylogenetic relationship between A. marginale isolates 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. marginale DNA was found in 89.7% of the examined zebu cattle and in 62.3% of the examined ticks. The tandem repeat and microsatellite analyses of the mspa1 gene showed high genetic diversity among the isolates between and within the different regions and high infection potential. Eighteen of the 25 tandem repeats identified have not been described before. Phylogenetic analysis revealed clustering of A. marginale strains from Madagascar with South Africa, America and Israel. A common ancestor may originate from South Africa and may have evolved due to phylogeographic characteristics or by a history of cattle movement. Its high prevalence in cattle and ticks, together with a low number of clinical manifestations and a high genetic heterogeneity among the investigated strains, confirms endemic stability of A. marginale in cattle from Madagascar.