Stem rust, caused by Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn., is one of the major diseases of wheat throughout the world. New P. graminis f. sp. tritici races including Ug99 (strain TTKS) and its variants, as well as local stem rust races, pose a serious threat to wheat (Triticum aestivum L.) production in Pakistan. Identifying resistance genes effective against the prevalent races and incorporating these genes into adapted wheat varieties can contribute to stem rust control. In this study, 117 Pakistani wheat varieties were screened with 18 DNA markers to detect the presence of stem rust resistance genes Sr2, Sr6, Sr22, Sr24, Sr25, Sr26, Sr31, and Sr38. Stem rust resistance genes Sr22, Sr24, Sr25, and Sr26 were absent from all varieties, whereas Sr2, Sr6, Sr31, and Sr38 were present at various frequencies. The highest frequency was observed for Sr2 (9–79% by different markers), followed by Sr31 (35%), Sr6 (11%), and Sr38 (9%). These results indicated that Pakistani varieties are being protected by very few resistance genes and lack resistance genes potentially effective against new stem rust races. Therefore, there is a need to incorporate stem rust resistance genes Sr22, Sr24, Sr25, and Sr26 into Pakistani wheat varieties. Different markers used for adult plant resistance gene Sr2 indicated different frequencies of this gene in the tested varieties. More reliable and efficient markers need to be developed for marker-assisted selection of this and other genes.