Puccinia striiformis f. sp. tritici (Pst) is the obligate biotrophic fungus responsible for stripe rust wheat. In this study, we developed and characterized 20 polymorphic microsatellite markers from the genomic sequence of an isolate of Chinese Pst race CY32. Polymorphism at each simple sequence repeat (SSR) locus was determined using 32 Pst isolates from 7 countries. The number of alleles varied from 2 to 7 across isolates, and the observed and expected heterozygosities ranged from 0.33 to 0.97 (mean 0.62) and 0.23 to 0.73 (mean 0.51), respectively. As expected the genomic SSR markers were more polymorphic than the expressed sequence tag (EST)-SSR markers developed previously. These markers will be more useful for population genetics and molecular genetics studies in Pst.