Background Brucellosis, as a common zoonotic disease from Iran, is a particularly essential animal disease that is occasionally observed in horses (1,2). Livestock plays an important role in supplying milk, meat, and wool in Iran. Additionally, horses are used as farmworkers, and for transport, equitation, and sports in rural and suburb areas of urban regions. Therefore, they play a significant role in the distribution of infection in animal to animal and/ or animal to human models (3). Three species of Brucella (i.e., B. abortus, B. suis, and B. canis) are the causes of brucellosis in the horse but B. abortus is the dominant species (4). Genitally tract disorders and reproductive failures such as abortion and stillbirth are the routine findings of infections in animals. Clinical symptoms in horses are termed as "poll-evil" or "fistulous withers", leading to abscess and fistulation in the affected location (5). The isolation of bacteria is a gold standard for diagnosis although techniques based on immuno-serology such as Rose-Bengal plate test (RBPT) and standard tube agglutination test (STAT) are user friendly because of rapid function and low cost (2,5). There are 1750 horse populations in the equestrian clubs, breeding farms, and rural regions of Hamedan province. According to the Iranian Veterinary Organization (IVO) approach, there is no vaccination program against brucellosis in horses (1). Brucellosis in horses from Iran and other countries (Figure 1) was reported in limit scales (3,6-11). To compensate for this fracture, the current project was designed to screen Brucella infection in the horses of the region based on serology. Methods In a cross-sectional study conducted during April 2019-January 2019, 495 blood samples (5 mL from the jugular vein) of the horses were collected from different equestrian clubs (n = 24) in Bahar, Razan, Asadabad, and Hamedan in Hamedan province (Figure 1). The animals were of different breeds without the clinical signs of disease. The samples were taken from the animals after obtaining official permissions and under the supervision of the Institutional Animal Ethics and Research Committee of IVO (Certificate No. 98/3127/27838). After the preparation of sera samples (centrifuge at 1200 ×g for 12 minutes), they were screened for the presence of anti-Brucella antibodies using the RBPT. Briefly, equal volumes (30 μL) of RBPT antigens (Razi Vaccine and Serum Institute, Iran) and serum samples were placed on a white ceramic tile and then mixed and rocked gently for 4 minutes. The reaction was recorded positive when the agglutination was observed and the antigens could detect B. abortus, B. melitensis, and B. suis (12). Results and Discussion Background on the prevalence and risk factors associated with brucellosis in animals is extremely important because of the zoonotic impact (1). In our serology