Stem rust, caused by the basidiomycetous fungus Puccinia graminis f. sp. tritici (Pgt), is one of the most devastating wheat diseases worldwide. Despite identifying many stem rust resistance genes (Sr) in wheat, efficacy of these genes is gradually being affected due to occurrence of virulent Pgt races. Selection pressure on Pgt populations due to planting of wheat cultivars with only one effective Sr gene, as well as existence of local Barberry shrubs needed for sexual reproduction of the pathogen, could result in emergence of new virulence to Sr resistance genes. This paper describes new virulence combinations on some Sr genes based on virulence assay of 123 Iranian P. graminis f. sp. tritici isolates collected during 2014-16. Purified urediniospore isolates were individually inoculated on 20 North American as well as 45 supplementary differential wheat cultivars carrying different Sr resistance gene/s. The Stakman et al. 0-4 scale was used for visual evaluation of infection types (IT) on seedling leaves at 14 days post inoculation (dpi). Our results showed virulence frequencies to stem rust resistance genes Sr22, Sr32, Sr33, Sr35, Sr39 and Sr40 at 35%, 15%, 40%, 80%, 5% and 14%, respectively. Various virulence frequencies were observed to other Sr genes ranging from 45 to 100%, indicating a high pathogenic diversity among Pgt populations in Iran. Utilization of these data in wheat breeding programs could result in new resistant cultivars carrying effective Sr genes in different stem rust infected areas.