BACKGROUND: Brucellosis is one of the most dangerous worldwide infectious zoonotic diseases that are common between ruminants and human. Consumption of infected milk and by-products is the major transmission source to human. In Iran, sheep compared to cow, has a higher rate of contamination with brucellosis. Therefore, early detection and precision could be a starting point for any efficient program to control the disease in human and animals. For brucellosis monitoring, milk ring test (MRT) is recommended but the test is not reliable in sheep herds. Perhaps a more realistic outcome could be achieved by changing the antigen used in MRT. OBJECTIVES: Comparison of two Brucella abortus and Brucella melitensis antigens in MRT for detection of Brucella antibodies in milk, as well as monitoring contamination of ewe’ s milk in Dezful region by detection of B. abortus and B. melitensis genes using PCR. METHODS: In this research, 220 milk samples from 16 different herds were collected from Dezful region’ s nomadic at Khuzestan province. As the first step, MRT by two antigens, B. abortus and B. melitensis, were conducted on the samples. Next, the samples were subjected to detect Brucella genes using PCR technique. RESULTS: Results showed that 47 (21/3 %) out of 220 cases were positive by MRT test, in terms of both antigens of B. abortus and B. melitensis. In PCR, out of 220 samples, only 9 (4%) samples were positive for specific genes of B. melitensis which were MRT positive as well. CONCLUSIONS: A significant difference between B. abortus and B. melitensis antigens was not observed in MRT. Although the nature and basis of PCR and MRT methods for the diagnosis of brucellosis is different but a significant difference between the results obtained by PCR and MRT showed that MRT even by changing of antigens is still not authentic. Considering that various methods of identification have their limitations, it is recommended that in ewe’ s milk samples, in addition to using a serological method as screening, PCR and culture methods should be used for definitive diagnosis.