ABSTRACT Mildew resistance Locus O (MLO), a gene family specific to plants, plays significant roles in the resistance to powdery mildew (PM) and response to a variety of abiotic stresses, plant growth and development. Despite their importance as barley, rice, wheat, few studies are reported in dicots except Arabidopsis; no global analysis has been performed in the burgeoning model fruit plant sweet orange (Citrus sinensis). The recent release of the genome sequences of C. sinensis provides an opportunity to conduct a comprehensive overview the evolution and features of the MLO gene family in sweet orange. In this study, amount to 14 members of the Citrus sinensis MLO gene (CisMLO) family according to their gene structures, conserved motifs, and similitude among their presumptive Arabidopsis and rice orthologs were identified in silico. Based on these analyses, all CisMLOs were grouped into six clades and expanded partly due to one tandem duplication and two segmental duplication events. Survey of their chromosomal distributions uncovered that 14 CisMLOs are localized across 6 chromosomes. Multiple-sequence alignments showed that 11 of them shared seven highly conserved transmembrane domains (TMs), while all of the sweet orange MLO proteins except CisMLO4/14 had a calmodulin-binding domain for MLO function. Expression analysis demonstrated that the MLO gene family has a diverse tissue-specific expression profiles in the sweet orange development and plays potential critical roles in stress responses. These findings will facilitate further studies of evolutionary pattern and biological functions of MLO genes in sweet orange.