Yellow rust (Puccinia striiformis f. sp. tritici, Pst) is a potentially dangerous disease of wheat. Genetic protection of wheat is an environmentally safe method of control. For its successful application, information on the structure of regional populations of the pathogen is needed. The purpose of these studies is to characterize the virulence and molecular polymorphism of Russian Pst populations in 2022. Wheat leaves with Pst urediniopustules were obtained from the North Caucasus (Krasnodar Territory, Dagestan, Kalmykia) and Northwestern (Leningrad region) regions. Virulence analysis was performed on 14 isogenic lines (AvocetNIL) and 15 differentiator varieties. The polymorphism of 20 microsatellite loci was evaluated in molecular analysis. We used SSR markers recommended by the Global Rust Reference Center. Virulence was studied in 74 monopustular isolates: 29 Dagestan, 10 Krasnodar, 5 Kalmyk, 30 northwestern. Resistance to all isolates was shown for lines with genes Yr5, Yr10, Yr15, Yr24, Yr26 and varieties Moro (Yr10, YrMor) and Nord Desprez (Yr3, YrND, Yr+). Isolates virulent to the AvYr17 line were detected for the first time in Dagestan and Krasnodar populations. They were moderately represented in the northwestern population (13%). Significant variation was observed in lines and varieties with the Yr1 and Yr3 genes. There was a decrease in virulence to Yr7 and YrSp compared to 2019–2021. 28 phenotypes (races) were determined in the virulence analysis (15 in Dagestan, 11 in North West, 3 in Krasnodar and 2 in Kalmyk). A common phenotype was noted for three North Caucasian Pst samples. The genetic distances between the identified phenotypes were estimated. In the multidimensional diagram, most of them combined together in a common group, with the exception of three Dagestan phenotypes with the fewest virulence alleles. According to the Fst index, Dagestan and Kalmyk Pst collections were characterized by high similarity; others differed moderately from them. The long-term virulence dynamics (2019–2022) of Pst populations in the Northwestern and North Caucasian regions was assessed. High similarity was determined between all regional population samples in 2019 and 2020. The northwestern and Dagestan populations differed slightly from them in 2021 and 2022. In 2022, the Krasnodar and Kalmyk populations were divided into separate groups, which differed from each other and from the main group. Long-term results of virulence analysis indicate a high dynamic of the structure of Pst populations in Russia. All North Caucasian isolates and 23 Northwestern isolates were used in the SSR analysis. Six loci (RYN3, RYN9, RYN12, WU6, RJO21, RJO24) were monomorphic. Three polymorphic alleles were identified in the RYN13 and RJO27 loci and two in the remaining studied ones. Significant deviations from the Hardy-Weinberg equilibrium are noted for most loci. The observed heterozygosity exceeded the expected one, which indicates an excess of heterozygotes and the clonal origin of the population. The studied collection of isolates was represented by 20 multilocus genotypes (MGs) (Dagestan and Northwestern – 11 each, Krasnodar – 3, Kalmyk – 1). Common genotypes were detected in Dagestan, Krasnodar and Northwestern populations (MG_1); Dagestan, Kalmyk and Northwestern (MG_2); Dagestan and Krasnodar (MG_3, MG_4). The genetic distances between MGs were estimated. In the multidimensional diagram, they are divided into 4 groups. The main group included 80% of MGs. One Dagestan MG, two Northwestern MG and MG_3 common for Dagestan and Krasnodar collections, significantly differentiated from the main group and among themselves. According to the Fst index, most regional Pst collections were moderately differentiated among themselves, with the exception of Dagestan and Kalmyk, which is consistent with the virulence analysis. According to the Mantel test, a moderate correlation was found between the results of virulence analysis and SSR (r = 0.6). This indicates that both analyses can be used in assessing the genetic polymorphism of Pst. The high variability of Russian populations based on virulence and microsatellite loci determines the need for annual monitoring of regional Pst populations in Russia.