Wheat production in Pakistan is seriously constrained due to rust diseases and stripe rust (yellow) caused by Puccinia striiformis f.sp.tritici,which could limit yields.Thus development and cultivation of genetically diverse and resistant varieties is the most sustainable solution to overcome these diseases.The first objective of the present study was to evaluate 100 Pakistan wheat cultivars that have been grown over the past 60 years.These cultivars were inoculated at the seedling stage with two virulent stripe rust isolates from the United States and two from Pakistan.None of the wheat cultivars were resistant to all tested stripe rust isolates,and 16% of cultivars were susceptible to the four isolates at the seedling stage.The data indicated that none of the Pakistan wheat cultivars contained either Yr5 or Yr15 genes that were considered to be effective against most P.striiformis f.sp.tritici isolates from around the world.Several Pakistan wheat cultivars may have gene Yr10,which is effective against isolate PST-127 but ineffective against PST-116.It is also possible that these cultivars may have other previously unidentified genes or gene combinations.The second objective was to evaluate the 100 Pakistan wheat cultivars for stripe rust resistance during natural epidemics in Pakistan and Washington State,USA.It was found that a higher frequency of resistance was present under field conditions compared with greenhouse conditions.Thirty genotypes (30% of germplasms) were found to have a potentially high temperature adult plant (HTAP) resistance.The third objective was to determine the genetic diversity in Pakistan wheat germplasms using molecular markers.This study was based on DNA fingerprinting using resistance gene analog polymorphism (RGAP) marker analysis.The highest polymorphism detected with RGAP primer pairs was 40%,50% and 57% with a mean polymorphism of 36%.A total of 22 RGAP markers were obtained in this study.RGAP,simple sequence repeat (SSR) and sequence tagged site (STS) markers were used to determine the presence and absence of some important stripe rust resistance genes,such as Yr5,Yr8,Yr9,Yr15 and Yr18.Of the 60 cultivars analyzed,17% of cultivars showed a RGAP marker band for Yr9 and 12% of cultivars exhibited the Yr18 marker band.No marker band was detected for Yr5,Yr8 and Yr15,indicating a likely absence of these genes in the tested Pakistan wheat cultivars.Cluster analysis based on molecular and stripe rust reaction data is useful in identifying considerable genetic diversity among Pakistan wheat cultivars.The resistant germplasms identified with 22 RGAP markers and from the resistance evaluations should be useful in developing new wheat cultivars with stripe rust resistance. Wheat production in Pakistan is seriously constrained due to rust diseases and stripe rust (yellow) caused by Puccinia striiformis f.sp.tritici, which could limit yields.Thus development and cultivation of genetically diverse diverse and resistant varieties is the most sustainable solution to overcome these diseases.The first objective of the present study was to evaluate 100 Pakistan wheat cultivars that have been grown over the past 60 years. These cultivars were inoculated at the seedling stage with two virulent stripe rust isolates from the United States and two from Pakistan. None of the wheat cultivars were resistant to all tested stripe rust isolates, and 16% of cultivars were susceptible to the four isolates at the seedling stage. the data indicated that none of the Pakistan wheat cultivars contained either Yr5 or Yr15 genes that were considered to be effective against most P.striiformis f.sp.tritici isolates from around the world.Several Pakistan wheat cultivars may have gene Yr10, which is effective against isolate PST-127 but ineffective against PST-116. It is also possible that those previously previously unidentified genes or gene combinations. second objective was to evaluate the 100 Pakistan wheat cultivars for stripe rust resistance during natural epidemics in Pakistan and Washington State, USA. It was found that a higher frequency of resistance was present under field conditions compared with greenhouse conditions. Thirty genotypes (30% of germplasms) were found to have a potentially high temperature adult plant (HTAP) resistance. objective was to determine the genetic diversity in Pakistan wheat germplasms using molecular markers. This study was based on DNA fingerprinting using resistance gene analog polymorphism (RGAP) marker analysis. The highest polymorphism detected with RGAP primer pairs was 40%, 50% and 57% with a mean polymorphism of 36%. A total of 22 RGAP markers were obtained in this study. RGAP, simple sequence repeat (SSR) and sequenOf the 60 cultivars analyzed, 17% of cultivars showed a RGAP marker band (STS) markers were used to determine the presence and absence of some important stripe rust resistance genes, such as Yr5, Yr8, Yr9, Yr15 and Yr18. for Yr9 and 12% of cultivars exhibited the Yr18 marker band. No marker band was detected for Yr5, Yr8 and Yr15, indicating a likely absence of these genes in the tested Pakistan wheat cultivars. Cluster analysis based on molecular and stripe rust reaction data is useful in identifyingfficient genetic diversity among Pakistan wheat cultivars. The resistant germplasms identified with 22 RGAP markers and from the resistance evaluations should be useful in developing new wheat cultivars with stripe rust resistance.