The southern region of Gansu province, including Tianshui city and Longnan city, is a hot and the first emergency area of Puccinia striiformis f.sp. tritici (Pst) in China. Stripe rust is the most destructive disease of wheat in this area. Deployment of resistant-conferring wheat varieties is the most economical, environmental-friendly and effective method. Five wheat germplasm accessions including Guinong 19, Guinong 21, Guinong 22, Guinong 29 and Guinong 775 were derived from Guizhou university. Since 1994, these accessions have been widely used as parental lines in breeding for stripe rust resistance wheat cultivars in this area. We have conducted a long-term experiment during 1999-2020, in order to test for resistance to stripe rust at the seedling and adult stage both under greenhouse and field conditions. Three lines Guinong 19, Guinong 21, Guinong 22 have become susceptible by infection with Pst isolate CYR34, which was the ranking first popular race in Gansu province since 2015. Two accessions Guinong 29 and Guinong 775 remain to be resistant to CYR34 , and both are valuable in wheat breeding in southern region of Gansu province in future.