Abstract Background Tick-borne diseases caused by Anaplasma species put serious constraints on the health and production of domestic cattle in tropical and sub-tropical regions. After recovering from a primary infection, cattle typically become persistent carriers of pathogens and play a critical role in the epidemiology of the disease, acting as reservoirs of the Anaplasma spp. Methods In this study a duplex PCR assay was used for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle using two primer pairs targeting msp 4 and msp 2 genes, respectively. We used this method to analyze DNA preparations derived from 328 blood cattle samples that were collected from 80 farms distributed among Tunisia’s four bioclimatic zones. Results The prevalence of the A. marginale infection (24.7 %) was significantly higher and more widespread (in all bioclimatic areas) than that of A. phagocytophilum (0.6 %), which was found in a mixed infection with A. marginale . Conclusions The duplex PCR assay used proved to be a rapid, specific and inexpensive mean for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle blood. It allowed us to report the identification of A. phagocytophilum for the first time in cattle in Tunisia and confirm the presence of A. marginale in cattle from several geographical areas of the country. Further epidemiological studies undertaken using this assay will help improve the surveillance of the associated diseases in the regions where they are endemic.