Anaplasmosis is a severe tickborne disease of ruminants caused by Anaplasma marginale. A. marginale is 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 characterize A. marginale isolated 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 of Anaplasmataceae, specifically A. marginale. The animals varied in breed, age, and gender, with most showing no signs of severe disease. By species, A. marginale was found in 61 out of 100 (61%) cattle, 9 out of 75 (12%) buffaloes, and only 5 out of 75 (6.66%) camels. All A. marginale-positive samples were examined for the heat-shock protein groEL gene and, additionally, for major surface protein 4 (msp4) and major surface protein 5 (msp5) genes to enhance specificity. Phylogenetic analysis of A. marginale targeted three genes (groEL, msp4, and msp5). This study provides the first report on using three genes for A. marginale detection in Camelus dromedarius in southern Egypt and generated new phylogenetic data for A. marginale infections in camels. A. marginale infection is endemic in different animal species in southern Egypt. Screening herds for A. marginale is recommended even when the signs of anaplasmosis are absent.