The appearance and spread of races of Puccinia graminis f. sp. tritici with virulence for the Sr31 resistance gene has renewed interest in breeding for durable resistance to stem rust of wheat. Since the occurrence of stem rust has been low in South Africa until the detection of race TTKSF in 2000, breeding for resistance to this disease has not been a primary objective. The aim of this study was to test bread wheat cultivars and lines at the seedling stage for their infection response to stem rust, thus determining their level of resistance or vulnerability. A collection of 65 bread wheat entries was tested with one USA race, two Eastern African races, and three South African races of P. graminis f. sp. tritici. The Eastern African and South African races all belong to the Ug99 lineage. The cultivars Duzi, Caledon, Elands, PAN 3364, PAN 3191, SST 047, SST 399, and Steenbras produced resistant infection types (IT < 3) to all races. The molecular marker Sr24#50 indicated the presence of Sr24 in 12 entries. In cultivars resistant to TTTTF, TTKSF, and TTKSP but susceptible to TTKSK and PTKST, the iag95 DNA marker indicated the presence of Sr31 in five wheat lines. Using the cleaved amplified polymorphic sequence marker csSr2, Sr2 was detected in PAN 3377, Inia, and Steenbras. Few South African wheat cultivars appear to have a broad-based resistance to stem rust, as 88% of the entries were susceptible as seedlings to at least one of the races tested. Diversification of resistance sources and more directed breeding for stem rust resistance are needed in South Africa.