This study aimed to test advanced wheat lines for resistance to stripe rust before releasing as new cultivars. Seedlings of 1,152 wheat lines were evaluated with three Pakistani isolates PK07-4, PK07-12 and PK08-2 representing races of Puccinia striiformis f. sp. tritici under controlled greenhouse conditions at Washington State University, Pullman, WA, USA. All three isolates are avirulent to resistance genes Yr5, Yr15 and YrSP, but isolates PK07-4 and PK07-12 are also avirulent to Yr10. Of the tested wheat lines, 114 were resistant to the three isolates. These genotypes may have Yr5, Yr15, YrSP and/or other genes effective against these three isolates. Resistant genes Yr24 and/or Yr32 were postulated in 189 lines which showed resistance to isolate PK07-4 which is avirulent to these genes. Similarly, 258 lines were resistant to isolate PK07-12 indicating possible presence of YrTr1 which is effective against this isolate. Another 19 genotypes showed resistant to both PK07-4 and PK07-12, but not PK08-2, indicating possible Yr10 in these lines. It is also possible that these lines may have other previously unidentified genes or gene combinations. The results of resistance in the wheat lines shared with national breeding programs facilitated releases of new wheat cultivars and further improvement of breeding materials for resistance to stripe rust.