Background Brucellosis is one of the most well-known and important zoonoses in the world, especially in Iran. Horse milk is a rare and expensive product with high nutrient ingredients in comparison to cow milk. In current study, a total of 164 blood and milk samples were randomly selected from 82 mares in Yazd city, Iran. Rose Bengal test (RBT) and milk ring test (MRT) methods were used as serological methods. A genus-specific PCR test targeting IS711 was performed using the primers ISP1 and ISP2 to identify Brucella. Specific primers were also used to identify Brucella abortus and Brucella melitensis at species level and then samples were sequenced. Positive PCR samples were cultured for bacterial isolation. Results Based on serological tests in milk and blood samples, no positive case was found. None of the blood samples were positive to identify Brucella genus, but 3 milk samples (3.66%) were positive in PCR assay. All 3 samples were identified as Brucella abortus. However, the microbiological culture of these samples did not lead into isolation of bacteria and no growth was observed. PCR can be applied as a sensitive method for diagnosis of Brucella infection in horses. Conclusion As the potential of Brucella infection confirmed by PCR in current study, special considerations should be taken to prevent close contact between horses and natural. Brucella hosts particularly in regions where horse milk is consumed by people. Health authorities should be aware of possible transmission of brucellosis by horse milk consumption which should be considered in control measures.