MLO, a plant-specific disease resistance gene family, was also found to play important roles in responses to abiotic stress. To explore their functions in foxtail genome, 12 SiMLO genes were identified through bioinformatics. The characterization of protein structure, sub-cellular location, phylogeny, conservative amino acid residues and gene expression were systematically analyzed.Structural analysis revealed that these proteins have one conserved MLO domain and multiple transmembrane domains, and conserved residues and motifs are also existed in these proteins. The majority of SiMLO proteins are located in plasma membrane. Phylogenetic analysis of MLO genes from foxtail and other species divided them into seven clades (Ⅰ~Ⅶ). All members from clade Ⅶ specifically belong to the family of Gramineae, suggesting that there exists a separate evolution process among these genes. SiMLO10 grouped into clade Ⅳ comtaining powdery mildrew resistance genes, show this gene with potential function in disease resistance. All known functional MLO genes from monocots and dicots were classed into clades Ⅳ and Ⅴ, respectively, indicating that the divergence of monocots and dicots preceded the formation of MLO function. Foxtail millet RNA-seq data unraveled that MLO genes have distinct expression patters in different tissues, suggesting these genes probably have distinct functions. These results will lay a foundation for further understanding the evolution,functions of MLO genes, as well as breeding new foxtail millet varieties resistant to powdery mildew disease.