Puccinia striiformis f. sp. tritici (Pst) is a destructive pathogen that causes wheat stripe rust worldwide. Understanding the population structure and dynamic of pathogen spread is critical to fight against this disease. Limited information is available for the population genetic structure of Pst in Uzbekistan, Central Asia. In this study, we carried out surveillance from 9 different regions (Andijan, Fergana, Jizzakh, Kashkadarya, Namangan, Samarkand, Sirdaryo, Surkhandarya and Tashkent) of Uzbekistan to fill this gap. A total of 255 isolates were collected, which were genotyped using 17 polymorphic simple sequence repeats (SSR) markers. The DAPC analysis results showed no population subdivision in these sample-collected regions except Surkhandarya. Multilocus genotype (MLG) analysis, FST, and Nei’s genetic distance results indicated a clonal population (rBarD ≤ 0.12) and merely three MLGs accounting for 70% of the overall population. MLG-34 was predominant in all Uzbekistan regions, followed by MLG-36 and MLG-42. Low genotypic diversity was observed in Andijan, Fergana, Jizzakh, Kashkadarya, Namangan, Sirdaryo, and Tashkent (0.56 to 0.76), compared with Samarkand (0.82) and Surkhandarya (0.97). No virulence against Yr5, Yr15, YrSp, and Yr26 was found, while resistant was overcome against Yr1, Yr2, Yr6, Yr9, Yr17, and Yr44 genes (Virulence frequency =≥75%). Comparative study results of Uzbekistan with previous Himalayan population were showed divergence from China and Pakistan populations. Further studies need to be conducted in a worldwide context to understand migration patterns; for that purpose, collaborative work is essential due to the Pst long-distance migration capability.