Isolates of Puccinia triticina collected from durum wheat from Argentina, Chile, Ethiopia, France, Mexico, Spain, and the United States were analyzed with 11 simple sequence repeat (SSR) markers in order to determine the genetic relationship among isolates. These isolates also were compared with P. triticina isolates from common wheat from North America, and an isolate collected from Aegilops speltoides from Israel, to determine genetic relationships among groups of P. triticina found on different telial hosts. The large majority of isolates from durum wheat were identical for SSR markers or had <8% genetic dissimilarity, except for isolates from Ethiopia, which had 55% dissimilarity with respect to the other durum isolates. Isolates from common wheat had >70% genetic dissimilarity from isolates from durum wheat, and the isolate from A. speltoides was >90% dissimilar from all isolates tested. Analysis of molecular variance tests showed significant levels (P = 0.001) of genetic differentiation among regions and among isolates within countries. Isolates of P. triticina from durum wheat from South America, North America, and Europe were closely related based on SSR genotypes, suggesting a recent common ancestor, whereas P. triticina from Ethiopia, common wheat, and A. speltoides each had distinct SSR genotypes, which suggested different origins.