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In Iraq, no data are available about the prevalence of Theileria annulata in buffaloes. For this reason, 184 blood samples were collected from buffaloes of different areas in Maysan province (Iraq) during June and September (2020), to be subjected for microscopic and serological testing using ELISA. The findings revealed that the total positive animals were 6.52% by microscopy and 16.85% by ELISA. Among seropositives, mild infection was increased significantly when compared to moderate and severe infections. Concerning sex factor, no significant differences were detected between females and males by microscopy, however, ELISA were revealed a significant increase in positive females compared to males. In seropositive females, there were significant increases in values of ELISA in comparison to those reported by light microscopy; while in males, no significant variation was showed between values of microscopy and those of ELISA. Regarding age factor, significant higher seropositives were reported in buffaloes aged ≥ 2- < 6 years by microscopy and ELISA when compared to buffaloes aged < 2 years and ≥ 6 years. Significantly, higher rates of prevalence were reported among all age’s groups by ELISA when compared to microscopic results. In conclusion, this appears to be the first serological study concerned serological investigation of T. annulata in buffaloes in Iraq. Therefore, additional studies using advanced diagnostic techniques are of great importance to detect the actual prevalence of infection among buffaloes of other Iraqi provinces.