Microsatellite analysis of ten South African races of Puccinia triticina, the causal organism of leaf rust of wheat (Triticum aestivum), divided the population into two groups sharing 32% genetic similarity. In the first group, race 3SA145 (Agricultural Research Council notation) clustered with 3SA122 and 3SA125, two races that have not been detected recently. This grouping was supported by avirulence for Lr1 in all three races. The second group included the current dominant race 3SA133, that shared 42% genetic homology with the remaining six races. Five of these, including 3SA144, formed a sub-group with 96% genetic homology amongst them. It was concluded that 3SA144, detected on triticale in 2005 and virulent for Lr32, probably represents a mutation from an existing race, whereas 3SA145 most likely was an exotic introduction. Race 3SA145 was first detected in 2009 and differed from known races in terms of combined virulence for the adult-plant resistance genes Lr12, Lr13 and Lr37. Microsatellite analysis complemented traditional phenotypic characterization of P. triticina and provided insight on genetic relationships amongst older and more recently detected races in South Africa.