A collection of Puccinia triticina isolates was characterized for polymorphism of microsatellite loci and estimated for their differentiation by geographic origin. The collection included 20 isolates from the Ural region, 31 from West Siberia, 53 from Central Europe, 32 from the Northwest region, 32 from the Volga region, and 40 from the North Caucasus (24 from Dagestan and 16 from Krasnodar and Stavropol). The studied isolates were represented by 65 virulence phenotypes. In the polymorphism analysis of 18 microsatellite loci, 69 genotypes were determined. The index values of genetic distances (Fst, Rst, KBc) between populations for microsatellite loci indicated differentiation of P. triticina isolates according to geographical origin, and they were clustered into three groups: (1) Asian, (2) European, and (3) North Caucasian. The North Caucasian isolates from Krasnodar and Stavropol regions were closer in similarity to European isolates than the Dagestan ones. Current analysis confirmed the assumption made earlier on the basis of the virulence analysis about the existence of several geographic fungi populations in Russia.