Several South African winter wheat (Triticum aestivum L.) varieties have been observed to express seedling resistance to local races of Puccinia graminis f. sp. tritici (Pgt). To study inheritance of seedling resistance the cultivars Komati, Koonap, Limpopo and SST 387 were crossed with the stem rust susceptible wheat Line 37-07. Using Pgt race PTKST the segregation of resistance in each of the four F2 populations indicated the presence of a single gene. Allelism tests conducted among these resistant parents revealed resistant F2 progeny only, suggesting that either the same gene or closely linked alleles confer resistance in the four wheat cultivars. The resistance gene mapped to the short arm of chromosome 6D and was flanked by microsatellite markers Wms4862 and Barc183 in all four populations. The winter wheat cultivars tested negative using diagnostic single nucleotide polymorphism (SNP) markers linked to Sr42, SrTmp and SrCad, all located on chromosome 6DS. Allelism tests between the resistant winter wheat cultivars and Norin40 (Sr42) and CnsSrTmp (SrTmp) were inconclusive as only resistant F2 progeny was obtained. This suggested that either an unknown, closely linked gene confers resistance to Pgt race PTKST in these cultivars or that Sr42 or SrTmp may indeed be involved. This research has provided evidence that the genetic base of stem rust resistance in a number of South African winter wheat varieties may be narrow.