Among the rust diseases, leaf rust of wheat caused by Puccinia triticina, is the most prevalent worldwide and causes significant yield losses. This study aimed to determine the genomic location of loci that control adult plant resistance (APR) to leaf rust in the pre-Green Revolution landrace accession, Aus27506, from the ‘Watkins Collection’. An Aus27506/Aus27229-derived F7 recombinant inbred (RIL) population was screened under field conditions across three cropping seasons and genotyped with the iSelect 90K Infinium SNP bead chip array. One QTL on each of chromosomes 1BL, 2B and 2DL explained most of the leaf rust response variation in the RIL population and were named QLr.sun-1BL, QLr.sun-2B and QLr.sun-2DL, respectively. QLr.sun-1BL and QLr.sun-2DL were contributed by Aus27506. QLr.sun-1BL is likely Lr46, while QLr.sun-2DL appeared to be a new APR locus. The alternate parent, Aus27229, carried the putatively new APR locus QLr.sun-2B. Comparisons of average severities among RILs carrying these QTL in different combinations indicated that QLr.sun-2B does not interact with either of the other two QTL; however, the combination of QLr.sun-1BL and QLr.sun-2DL reduced disease severity significantly. In-planta fungal quantification assays validated these results. The RILs carrying QLr.sun-1BL and QLr.sun-2DL did not differ significantly from parent Aus27506 in resistance. Aus27506 can be used as a source of adult plant leaf rust resistance in breeding programs.