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In recent years, wheat stem rust, caused by Puccinia graminis f.sp. tritici, has been reconsidered in Iran due to its prevalence and the emergence of the dangerous Ug99 race. This study was conducted to understand pathogenic variation in the population of P. graminis f.sp. tritici, detection of effective genes, and identification of resistance in Iranian commercial wheat cultivars or advanced lines, by planting stem rust trap nurseries under natural disease infection in several regions of Iran during the 2016-2017 cropping season. The trap nursery in each location included 48 wheat lines each carrying a single gene of stem rust (Sr) resistance, seven lines each carrying Sr multigenes, eight additional lines to confirm four Sr genes, 149 commercial wheat cultivars or advanced lines from Iran, plus several susceptible checks. The percentage leaf area affected (disease severity) and infection type were recorded at adult plant stage when disease was well developed on flag leaves of susceptible checks. Results showed presence of virulence for several Sr genes in one or more locations. However, the single genes of Sr13, Sr23, Sr24, and two complex genes of Sr7a+Sr6+Sr12 and Sr6+Sr24+Sr36+Sr1RS-Am were still effective against stem rust in all locations. The results of evaluations of commercial wheat cultivars or advanced lines showed that approximately 16% the genotypes tested including wheat cultivars Gonbad, Shiroudi, Chamran-2, Baharan, Dena, Karkheh, and Arya were resistant in all locations.