Brucella species are facultative intracellular pathogenic α-Proteobacteria that can cause brucellosis in humans and domestic animals. The clinical and veterinary importance of the bacteria has led to well established studies on the molecular mechanisms of Brucella infection of host organisms. However, to date, no genome-wide study has scanned for genes related to the host specificity of Brucella spp. The majority of bacterial genes related to specific environmental adaptations such as host specificity are well-known to have evolved under positive selection pressure. We thus detected signals of positive selection for individual orthologous genes among Brucella genomes and identified genes related to host specificity. We first determined orthologous sets from seven completely sequenced Brucella genomes using the Reciprocal Best Hits (RBH). A maximum likelihood analysis based on the branch-site test was accomplished to examine the presence of positive selection signals, which was subsequently confirmed by phylogenetic analysis. Consequently, 12 out of 2,033 orthologous genes were positively selected by specific Brucella lineages, each of which belongs to a particular animal host. Extensive literature reviews revealed that half of these computationally identified genes are indeed involved in Brucella host specificity. We expect that this genome-wide approach based on positive selection may be reliably used to screen for genes related to environmental adaptation of a particular species and that it will provide a set of appropriate candidate genes.