Yellow rust disease caused by Puccinia striiformis f.sp. tritici is the most important biotic stress of wheat crop being responsible for reasonable yield loss in the epidemics years. Since 1990 several epidemics have occurred in Iran and causing the breakdown of widely utilized sources of resistance in wheat cultivars. In 1993 wheat yield loss due to stripe rust was estimated 1.5 million tones in Iran. In the spring of 2010 widespread of stripe rust was started from west and north west of country, and most old cultivated wheat cultivars in this region showed susceptibility reaction. In this study twenty-one isolates of Pst. were collected from different parts of Iran. Pathotyping was done on the standard set of wheat stripe rust in greenhouse condition. The infection types were assessed on a 0-9 scale 16 and 18 days after inoculation using a scale similar to that described by McNeal et al. (1971). Infection types(ITs) 7 to 9 were regarded as virulent (susceptible) and less than 7 are avirulent. Pathotypes 238E190A+,Yr27+ (from Dezful), 174E158A+,Yr27+ (from Brojerd) and pathotype 78E158A+,Yr27+ from Garakhil were more aggressive during this study. Virulence on plant with gene/s Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr25,Yr27, Yr32, YrND, YrSD and YrA were more common and detected in greenhouse condition, and virulence for plants with the gene YrSU and YrCV was limited. For genes Yr1, Yr4, Yr5, Yr10, Yr15 and YrSP virulence was not detected during 2018. Use of resistant cultivar is the best method to control the disease. Because of screening of wheat germplasm over last 15 years to pathotype of stripe rust with virulence on Yr27, new released cultivars included Sirevan, Mehregan, Parsi, Sirvan, Baharan, Morvareid,Tirgan, Gonbad, Pishgam, Maihan, Haydarei and Shabrang were resistant to the new raising pathotype with virulence on plants with Yr27 in the field condition.