Ingestion of raw infected milk and dairy products is the most important way for human brucellosis. This study was carried out in order to investigate the prevalence of Brucella spp., Brucella abortus and Brucella melitensis in cow’s milk using culture, dot-ELISA and duplex PCR.  All of the 150 milk samples were cultured for Brucella spp. and the positive results have been evaluated for presences of Brucella abortus and Brucella melitensis using dot-ELISA and duplex PCR assays. Totally, 12% of milk samples were positive for Brucella spp. using culture. The dot-Elisa assay indicated that 12% and 2% of milk samples were positive for Brucella abortus and Brucella melitensis, respectively. Novel duplex PCR showed that 14%, 4% and 2% of milk samples were positive for Brucella abortus, Brucella melitensis and both bacteria. Isfahan had the highest incidence of Brucella spp. (17.5%), while Chaharmahal Va Bakhtiari had the lowest incidence of Brucella spp. (8%). There were significant differences about P < 0.05 for incidence of Brucella spp. between Isfahan with Chaharmahal Va Bakhtiari provinces. Our study showed that milk inspection should be performed in order to diminish the distribution of brucellosis in human populations.