Five thousand and one wheat varieties (lines) including four thousand two hundred and ninety-one winter wheat varieties (lines) and seven hundred and ten spring wheat varieties (lines) , which come from thirty-five breeding unite in China, were evaluated their resistance to stripe rust by artificial inoculated mixed tested races/isolates of puccinia striiformis f.sp tritici (Pst) at seedling stage in Lanzhou greenhouse, and tested single/mixed races/isolates of Pst at adult plant stage at Gangu testing station, institute of plant protection, Gansu academy of agricultural sciences (IPP GAAS) during 2003~2013. The results showed that four hundred and seventy-nine, seventy-six, two hundred and ninety-one wheat varieties (lines) were immune (IM), high resistance (HR) and middle resistance (MR) in seedling and adult plant stage to all tested single/mixed races/isolates of Pst, about 9.58%, 1.52% and 5.82%, respectively. Eight hundred and forty, forty-seven, three hundred and eleven wheat varieties (lines) were IM, HR and MR in adult plant stage, about 16.80%, 0.94% and 6.22%. respectively. Nine hundred and sixty-four, one hundred and twenty-four, two hundred and seventy-three wheat varieties (lines) were IM, HR and MR to tested mixed races/isolates of Pst in seedling stage, about19.28%, 2.44% and 5.46%. Nine hundred and fourteen, nine hundred and six, one thousand two hundred and twenty-five winter wheat varieties (lines) were resistance to all tested single/mixed races/isolates of Pst in all-stage, adult plant stage and seedling stage, about 18.28%, 18.12% and 24.50%, respectively. One hundred and thirteen, one hundred and twenty-five, one hundred and fourteen spring wheat varieties (lines) were resistance in all-stage, adult plant stage and seedling stage, about 2.26%, 2.50% and 2.28%, respectively. By natural induced isolates of Pst at Wangchuan farm to evaluated resistance in field during 2004~2013. The results showed that seven hundred and forty five wheat varieties (lines) which from races/isolates pool of Pst at Gangu testing station, IPP GAAS were resistance, about 64.56%. One hundred and five commercial wheat varieties in Gansu province, only thirty wheat varieties including Lantian 19, Tianxuan 44 were resistance in field from 2003 to 2013, about 28.57%. Some wheat lines of Gansu province especially including 00-30-2-1, CP04-20 and 00127-2-3, were immune or high resistance to stripe rust in field than other wheat lines. There were a few wheat germplasms including Guinong 775, Zhong 5, T.Spelta album, Guixie 1 and Guixie 3 were resistant to tested and natural races/isolates of Pst, and mostly importance to the sources including Guinong 21, Guinong 22, 92R, Chuanmai 42 and Moro were susceptible to stripe rust in field since 2011, and lost its value in planting and breeding. The offspring wheat varieties including Longjian 9343, Tianxuan 43, Zhongliang 29, Lantian 17 and Lantian 24, were susceptible slowly to stripe rust in field. Epidemic pressure continues to increase to stripe rust in field in Longnan region, Gansu province and China. Discussed the relationship between tested and natural inoculated isolates of Pst, and importance of using resistant wheat varieties (lines) in Longnan region, Gansu province in this paper.