The introduction of genes that confer resistance to major grapevine pests and pathogens into the highly susceptible V. vinifera cultivars used for wine production worldwide would be of signifi cant economic and environmental benefi t. The availability of genetic resources for grapevine has led to a dramatic increase in research on the mapping of resistance loci. Often the outcome of this work is the development of markers for marker-assisted selection of new disease-resistant interspecific hybrids. However, positional cloning of these resistance genes offers the possibility to introduce these genes into existing elite wine grape cultivars by grapevine transformation without affecting wine quality which has been a major obstacle in the adoption of hybrid vines generated by classical breeding. Furthermore positional cloning offers the possibility to introduce resistance genes into V. vinifera cultivars from species which cannot be achieved through hybridization. Finally, cloning of resistance genes provides sequence information that can be used to design perfect genetic markers which will maximize the effi ciency of marker assisted selection approaches. This chapter describes the strategy used, and technical challenges encountered, during the mapping and positional cloning of powdery mildew and downy mildew resistance genes from the wild North American grape species Muscadinia rotundifolia.