Powdery mildew (Pm) is one of the most harmful diseases in wheat. Three Pm-resistance genes, Pm3, Pm21, and Pm8, have been cloned but most Pm3/Pm8 alleles have lost their resistance to Pm in hexaploid wheat. In this study, a new Pm3 homolog gene (TmPm3) was isolated from Triticum monococcum L. using a homology-based cloning strategy, being the first report of a functional Pm3 homolog gene from a diploid wheat species. The transient expression of TmPm3 in leaf epidermal cells showed that over-expressed TmPm3 could significantly inhibit the penetration of Blumeria graminis f. sp tritici conidia spores and the formation of haustoria. Sequence analysis of Pm3 alleles shed new light on the evolution of Pm3 genes, providing a better understanding of the molecular basis of disease resistance. This study also suggested that homology-based cloning of resistance genes is a feasible method for the isolation of functional resistance genes from wheat germplasm.