Anaplasmosis is a severe tickborne disease of ruminants caused byAnaplasma marginale.A. marginaleis distributed worldwide and attacks erythrocytes, resulting in an increased body temperature, anemia, jaundice, abortion, and, in some cases, death. Animals infected with this pathogen become lifelong carriers. In this study, we aimed to detect and characterizeA. marginaleisolated from cattle, buffalo, and camel populations using novel molecular techniques in southern Egypt. In total, 250 samples (from 100 cattle, 75 water buffaloes, and 75 camels) were analyzed by PCR for the presence ofAnaplasmataceae, specificallyA. marginale. The animals varied in breed, age, and gender, with most showing no signs of severe disease. By species,A. marginalewas found in 61 out of 100 (61%) cattle, 9 out of 75 (12%) buffaloes, and only 5 out of 75 (6.66%) camels. AllA. marginale-positive samples were examined for the heat-shock proteingroELgene and, additionally, formajor surface protein 4(msp4) andmajor surface protein 5(msp5) genes to enhance specificity. Phylogenetic analysis ofA. marginaletargeted three genes (groEL, msp4, andmsp5). This study provides the first report on using three genes forA. marginaledetection inCamelus dromedariusin southern Egypt and generated new phylogenetic data forA. marginaleinfections in camels.A. marginale infectionis endemic in different animal species in southern Egypt. Screening herds forA. marginaleis recommended even when the signs of anaplasmosis are absent.