A systematic review of studies providing frequency estimates of brucellosis in humans and ruminants and risk factors for Brucella spp. seropositivity in humans in the Middle East was conducted to collate current knowledge of brucellosis in this region. Eight databases were searched for peer-reviewed original Arabic, English, French and Persian journal articles; the search was conducted on June 2014. Two reviewers evaluated articles for inclusion based on pre-defined criteria. Of 451 research articles, only 87 articles passed the screening process and provided bacteriological and serological evidence for brucellosis in all Middle Eastern countries. Brucella melitensis and B. abortus have been identified in most countries in the Middle East, supporting the notion of widespread presence of Brucella spp. especially B. melitensis across the region. Of the 87 articles, 49 were used to provide evidence of the presence of Brucella spp. but only 11 provided new knowledge on the frequency of brucellosis in humans and ruminants or on human risk factors for seropositivity and were deemed of sufficient quality. Small ruminant populations in the region show seroprevalence values that are among the highest worldwide. Human cases are likely to arise from subpopulations occupationally exposed to ruminants or from the consumption of unpasteurized dairy products. The Middle East is in need of well-designed observational studies that could generate reliable frequency estimates needed to assess the burden of disease and to inform disease control policies.