Stem rust is one of the major diseases of wheat round the world. New stem rust race; Ug99 and its variants as well as local stem rust races pose serious threat to wheat production in Pakistan. Stem rust can be controlled by identifying and incorporating resistant genes against the prevalent races into adapted wheat varieties. This study includes screening of 119 Pakistani wheat varieties with 18 DNA markers to detect the presence of various SR genes. Stem rust resistance genes Sr22, Sr24, Sr25 and Sr26 were absent in all varieties, whereas Sr2, Sr6, Sr9a, Sr31, Sr38 were present in various frequencies. However, frequency of APR gene Sr2 was quiet high. Results showed that Pakistani varieties are being protected by a few resistance genes and also lack potential resistance genes effective against new stem rust races. Thus, there is a need to incorporate stem rust resistance genes Sr22, Sr24, Sr25 and Sr26 into Pakistani wheat varieties, especially those possessing only Sr31. More reliable and efficient marker needs to be developed for Sr2 to assist MAS of this gene. Results of this study will help in pyramiding of SR genes in future wheat varieties to achieve durable rust resistance.