The wheat stripe/yellow rust (WYR) causing pathogen Puccinia striiformis f. sp. tritici (Pst) is one of the major biotic limitations in global wheat production and has the potential to cause major yield losses wherever wheat is grown. WYR can be successfully managed within and between cropping cycles by means of genetic resistance, reducing or even eliminating the need for fungicide intervention and resulting in increased productivity and a cleaner environment. To find new sources of resistance against Pst, a set of 766 hexaploid synthetic wheat lines were screened in artificially inoculated rust field nurseries for three seasons. A total of 591 lines were consistently resistant to WYR. From these 591 lines, a core set of 94 non-lodging lines with unique pedigrees was established. The core set was tested again in the field at adult plant growth stages, and as seedlings in greenhouse tests with six pathotypes of Pst that differed in virulence for the resistance genes YrA, Yr1, Yr2, Yr3, Yr4, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, Yr27, Yr32, Yr33 and YrJ. The lines were also genotyped with three molecular markers linked to adult plant resistance (APR) genes Yr18, Yr29 and Yr36. Under field conditions, the core set showed were categorised as being immune (5.5% of entries), highly resistant (45%), resistant (21%), moderately resistant (21%) and moderately susceptible (7.5%). Combined analysis of field rust responses, multi-pathotype phenotyping and genotyping revealed 34 classes of Pst resistance. Postulated resistance genes included Yr2 (2%), Yr6 (1%), Yr7 (1%), Yr8 (1%), Yr9 (20%), Yr17 (19%), Yr18 (35%), Yr25 (7%), Yr27 (14%), Yr29 (83%), Yr32 (26%), Yr33 (25%), unknown seedling resistance (38%) and unknown APR (18%), either singly or in combination. These lines represent a valuable resource for breeding stripe rust resistant varieties. Development of mapping populations has been initiated to characterize and map the unknown gene/s conferring resistance against Pst.