Background: Wheat powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici ( Bgt ), is a serious disease of wheat worldwide that can cause significant yield losses. Growing resistant cultivars is the most cost-effective and eco-soundly strategy to manage the disease.Therefore, a high breeding priority is to identify genes that can be readily used either singly or in combination for effective resistance to powdery mildew and alos in combination with genes for resistance to other diseases. Yunnan Province, with complex and diverse ecological environments and climates, is one of the main wheat growing regions in China. This region provides initial inoculum for starting epidemics of wheat powdery mildew in the region and other regions and thus, plays a key role in the regional and large-scale epidemics of the disease throughout China. The objectives of this study were to evaluate seedling resistance of 69 main wheat cultivars to powdery mildew and to determine the presence of resistance genes Pm3 , Pm8 , Pm13 , Pm16 , and Pm21 in these cultivars using gene specific DNA markers.Results: Evaluation of 69 wheat cultivars with six Bgt isolates showed that only four cultivars were resistant to all tested isolates, indicating that the overall level of powdery mildew resistance of Yunnan wheat cultivars is inadequate. The molecular marker results showed that 27 cultivars likely have at least one of these genes. Six cultivars were found likely to have Pm3 , 18 likely to have Pm8 , 5 likely to have Pm16 , and 3 likely to have Pm21 . No cultivar was found to carry Pm13 .Conclusion: The information on the presence of the Pm resistance genes in Yunnan wheat cultivars can be used in future wheat disease breeding programs. In particular, cultivars carrying Pm21 , which is effective against all Bgt races in China, should be pyramided with other effective genes to developing new cultivars with durable resistance to powdery mildew.