The deployment of diverse sources of resistance in new cultivars underpins durable control of rust diseases. Aus27430 exhibited a moderate level of stripe rust resistance against Puccinia striiformis f. sp. tritici (Pst) pathotypes currently prevalent in Australia. Aus27430 was crossed with the susceptible parent Avocet S (AvS) and subsequent filial generations were raised. Monogenic segregation observed among Aus27430/AvS F3 families was confirmed through stripe rust screening of an F6 recombinant inbred line (RIL) population, and the resistance locus was temporarily named YrAW5. Selective genotyping using an Illumina iSelect 90K wheat SNP bead chip array located YrAW5 in chromosome 6A. Genetic mapping of the RIL population with linked 90K SNPs that were converted into PCR-based marker assays, as well as SSR markers previously mapped to chromosome 6A, confirmed the chromosomal assignment for YrAW5. Comparative analysis of other stripe rust resistance genes located in chromosome 6A led to the formal designation of YrAW5 as Yr81. Tests with a marker linked with Yr18 also demonstrated the presence of this gene in Aus27430. Yr18 interacted with Yr81 to produce stripe rust responses lower than those produced by RILs carrying these genes individually. Although gwm459 showed higher recombination with Yr81 compared with the other flanking marker KASP_3077, it amplified the AvS allele in 80 cultivars, whereas KASP_3077 amplified AvS allele in 67 cultivars. Both markers can be used in marker-assisted selection after confirming parental polymorphism.