Brucellosis caused by Brucella melitensis is a serious public health problem in Palestine. This zoonotic disease is primarily affecting small ruminants however it can be transmitted to human causing a serious illness known as Malta fever. Brucella melitensis ‘Rev1’ is an attenuated strain that is widely used as a live vaccine to control Brucella melitensis infection in small ruminants. However, Rev1 vaccination can interfere with the diagnosis of Brucella melitensis. In addition, shedding of Rev1 in milk after vaccination or after abortion in pregnant animals can increase the risk for human infection. Despite the great need for an accurate assay to differentiate between the vaccinated and infected animals, Rev1 genotyping is still a challenging issue.