Abstract Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), poses a threat to wheat production in South Africa (SA). The locally developed winter wheat cultivar PAN 3161 has consistently displayed adult plant resistance (APR) to stem rust, prompting an investigation into the genetics thereof. An F2 population was developed from a cross between PAN 3161 and the susceptible parent Line 37 − 07 and tested against Pgt race PTKST in the field. F3 families were also assessed in the field to confirm the F2 segregation ratios. Molecular marker genotyping combined with bulk segregant analysis (BSA) on 128 F2 plants identified markers on the short arm of chromosome 4D to be linked to stem rust resistance. Simple sequence repeat (SSR) marker wmc720 flanked the APR gene, SrPan3161, distally at 1.8 cM. Five co-segregating SSR markers gpw7414, gpw8038, wmc52, psp3103 and cfd23 flanked SrPan3161 proximally at 1.8 cM. PAN 3161 tested negative for Lr67/Yr46/Sr55/Pm46/Ltn3, and linked (csSNP856) and diagnostic single nucleotide polymorphism (SNP) markers (SNP1-TM4 and SNP2-TM10). Quantitative trait loci (QTL) analysis was performed to confirm linkage mapping results and identified a single major QTL that explained 71.5% of the phenotypic variation for resistance to Pgt race PTKST. The closely linked SSR markers psp3103 and cfd23 were 100% predictive of SrPan3161 in a set of 55 wheat cultivars and breeding lines, thus validating chromosome 4D as the region carrying SrPan3161 and their potential effectiveness in marker-assisted selection. Results suggest that SrPan3161 was introduced into South African winter wheat through the cultivar Tugela (pedigree Kavkaz/Jaral), first released for production in 1985.