Isolates of Puccinia triticina collected from common wheat in the Central Asia countries of Kazakhstan, Uzbekistan, Tajikistan, and Kyrgyzstan and the Caucasus countries of Azerbaijan, Georgia, and Armenia were tested for virulence to 20 isolines of Thatcher wheat with different leaf rust resistance genes and molecular genotype at 23 simple sequence repeat (SSR) loci. After clone correction within each country, 99 isolates were analyzed for measures of population diversity, variation at single SSR loci, and for genetic differentiation of virulence phenotypes and SSR genotypes. Isolates from Central Asia and the Caucasus were also compared with 16 P. triticina isolates collected from common wheat in North America that were representative of the virulence and molecular variation in this region and two isolates collected from durum wheat in France and the United States. Populations from the Caucasus, Uzbekistan, Tajikistan, and Kyrgyzstan were not significantly (P > 0.05) differentiated for SSR variation with Fst and Rst statistics. Populations from the Caucasus, Uzbekistan, Tajikistan, and Kyrgyzstan were significantly (P < 0.05) differentiated from the populations in South and North Kazakhstan for SSR variation. All populations from Central Asia and the Caucasus were significantly differentiated from the North American isolates and isolates from durum wheat for SSR variation and virulence phenotypes. There was a correlation between virulence phenotype and SSR genotype among individual isolates and at the population level. Mountain barriers may account for the differentiation of P. triticina geographic populations in Central Asia and the Caucasus.