Background: Besnoitiosis caused by Besnoitia besnoiti is regarded as a re-emerging disease in cattle because of the increased number of cases and geographical distribution in many European countries. Aims: The present study was conducted to determine the presence of B. besnoiti in cattle in the Eastern and Southeastern Anatolia of Turkey. Methods: Blood samples were collected from 450 cattle in the provinces of Mus, Van, Siirt, and Diyarbakir. PrioCHECK (R) Besnoitia Ab 2.0 enzyme-linked immunosorbent assay (ELISA) kit was used to detect specific anti-B. besnoiti antibodies in the serum samples. Results: Twelve (2.7%) of the 450 asymptomatic cattle were seropositive against B. besnoiti. In cattle, the prevalence rates were 0%, 3.7%, 3.4%, and 1.1% in Mus, Siirt, Diyarbakir, and Van provinces (P>0.05), respectively. This study is the first to investigate the presence of B. besnoiti in cattle raised in the Eastern and Southeastern Anatolia of Turkey. Conclusion: Although the ELISA test revealed some positive cases, concrete evidence for the establishment of clinical B. besnoiti infection could not be verified. More comprehensive analysis would be necessary to determine the significance of the present observations.