The deployment of combinations of resistance genes in future wheat cultivars can save yield losses caused by the stripe rust pathogen (Puccinia striiformis f. sp. tritici; Pst). This relies on the availability and identification of genetically diverse sources of resistance. A Tunisian landrace Aus26670 displayed high level of stripe rust resistance against Australian Pst pathotypes. This landrace was crossed with a susceptible line Avocet ‘S’ (AvS) to generate 123 F₇ recombinant inbred lines (RILs). The Aus26670/AvS RIL population was evaluated against three Pst pathotypes individually in greenhouse and against mixture of Pst pathotypes under field conditions for three consecutive years. Genetic analysis of the seedling stripe rust response variation data indicated the presence of an all-stage resistance (ASR) gene, and it was named YrAW12. This gene is effective against Australian Pst pathotypes 110 E143A + and 134 E16A + Yr17 + Yr27 + and is ineffective against the pathotype 239 E237A-Yr17 + Yr33 + . The RIL population was genotyped using the targeted genotyping-by-sequencing (tGBS) assay. YrAW12 was mapped in the 754.9–763.9 Mb region of the physical map of Chinese Spring and was concluded to be previously identified stripe rust resistance gene Yr72. QTL analysis suggested the involvement of four genomic regions which were named: QYr.sun-1BL/Yr29, QYr.sun-5AL, QYr.sun-5BL and QYr.sun-6DS, in controlling stripe rust resistance in Aus26670. Comparison of genomic regions detected in this study with previously reported QTL indicated the uniqueness of QYr.sun-5AL (654.5 Mb) and QYr.sun-6DS (1.4 Mb). Detailed mapping of these genomic regions will lead to permanent designation of these loci.