Technical Abstract: Puccinia striiformis (Ps), the causal agent of stripe rust, is an obligate biotrophic fungus. The objective of this study was to identify polymorphic genes for determining the mechanisms of the pathogen variation. Primers were designed for seven important putative genes including beta-tubulin (BT), elongation factor (EF), mitogen-activated protein kinase (MAPK), TATA-box binding protein (TBP), serine-threonine kinase receptor-associated protein (STKRAP), conidiation protein (CP), and cell wall glucanase (CWG) selected from the full-length cDNA library of P. striiformis f. sp. tritici (Pst), the wheat stripe rust pathogen. The full-length genomic sequences of the seven genes were obtained for different race groups of Pst in the U.S. and China and P. striiformis f. sp. hordei (Psh), the barley stripe rust pathogen. The TBP and CP genes had identical sequences among all tested Ps races, but different from the stem rust homologues. The five remaining genes had various levels of polymorphism and therefore, were useful in determining the evolutionary relationships among isolates. The phylogenetic relationships for the races revealed that mutation is the major evolutionary mechanism to create genetic variation, and that genetic recombination, either somatic, sexual, or both, is possible, but not the major mechanism for the total identified polymorphism in this study. Of 120 polymorphic base pair sites detected in the five genes, 74 (62%) had heterokaryotic variations, indicating that heterokaryosis resulted from mutations and possibly somatic or sexual hybridization is very common in the population of the stripe rust pathogen. This is the first report of heterokaryotic variation at the gene sequence level for the Ps fungus.