Leaf rust, caused by Puccinia triticina, is the most prevalent and widespread disease of wheat in South America. The objective of this study was to determine whether genetically differentiated groups of P. triticina are currently present in South America and to compare the South American population with the previously characterized population in North America. In total, 130 isolates of P. triticina from the wheat-growing regions of Argentina, Brazil, Chile, Peru, and Uruguay, mostly from the 1990s to 2008, were tested for virulence on 20 lines of wheat with single genes for leaf rust resistance and for molecular genotypes with 23 simple-sequence repeat (SSR) markers. After removal of isolates with identical virulence and SSR genotypes, 99 isolates were included for further analysis. Principal coordinate analysis plots indicated five different groups of isolates based on SSR genotypes that also differed for virulence to leaf rust resistance genes. All pairs of groups, except for one pair, were significantly differentiated for SSR genotypes according to R(ST) statistics. All but two pairs of groups were significantly differentiated for virulence phenotype according to Phi(PT) statistics. Isolates in all five groups had high values of fixation index for SSR alleles and linkage disequilibrium was high across all isolates that indicated the clonal reproduction of urediniospores. Only one of the five P. triticina groups from South America was differentiated for SSR genotypes from all of the six P. triticina groups from North America. The high degree of similarity for SSR genotype of isolates from both South America and North America suggested a common European origin of P. triticina that was introduced to both continents. The emergence of the same P. triticina virulence phenotypes with highly related SSR genotypes in the United States in 1996 and in Uruguay in 1999 indicated the likely intercontinental migration of these genotypes from Mexico to both South America and North America.