STEM rust caused by Puccinia graminis f. sp. tritici is a destructive disease of wheat in Egypt and worldwide. Survey of wheat stem rust samples and identification of physiological races using twenty single Sr genes are very important in describing virulence pattern variation, the geographical distribution of stem rust pathotypes, and how its change in response to host selection. Variability in the population of the causal organism was determined using samples collected from wheat-growing areas in Egypt for two growing seasons, that is, 2015/2016 and 2016/2017. The results obtained showed significant variability in pathotypes, which are different from season to season. In the course of this study, a total of 104 and 40 stem rust samples were collected in 2015/2016 and 2016/2017, respectively from different wheat-growing areas in six governorates of Egypt, that is, Beheira, Kafr-Elsheikh, Sharqiya, Minufiya, Bani Sweif, and Sohag. A total of 70 and 53 physiologic races were isolated from samples collected in the previous growing seasons and identified in 2016/2017 and 2017/2018, respectively. The most frequent races included TKTTC (18.25%) and TTTTC (17.46%) in 2016/2017 as well as PKSTC (6.25%), BBBBC (4.69%) and PKSTH (4.69%) in 2017/2018. For 2016/2017, 24 pathotypes were identified in Kafr-Elsheikh, which consider the largest population size (34.28%), while for 2017/2018, the Sharqiya governorate considered to be the largest population size (49.05%). Cluster analysis based on percentage frequency of virulence of P. graminis race groups in different locations showed that in the 2016/2017 and 2017/2018 growing seasons, two main clusters were formed. Lines with Sr 24, Sr 38 and Sr 31 genes were showed the highest gene efficacy, while the other genes showed different reactions against the tested pathotypes.