One of the effective ways to control the damage of stripe rust in wheat is the introduction of stripe rust resistant genes into cultivars to improve their resistance. Molecular marker-assisted selection (MAS) can improve the selection efficiency of target genes in breeding. In order to detect the stripe rust resistant gene Yr69 and verify the validity of molecular marker linked to Yr69, the genotypes of 63 F6 lines derived from Chang 4738/CH7086 and Yannong 999/CH7086 were identified by molecular marker 2AS33,and their resistance to stripe rust were identified with the Pst mixed with CYR32, CYR32 and CYR34. The results showed that 32 lines among the 35 lines derived from Chang 4738/CH7086 had Yr69 and showed resistance to stripe rust, and one line showed resistance to stripe rust without Yr69 being detected; 8 lines among the 28 lines derived from Yannong 999/CH7086 had Yr69, of which 7 lines showed homozygous resistance and one line showed segregation of resistance and susceptibility. Among the total 63 lines, 3 had genotypes inconsistent with phenotypes. It suggested that the marker 2AS33 had a high accuracy of 95.2% and it could be used to detect the Yr69 gene effectively in molecular marker-assisted selection breeding. The 40 resistant lines identified, after yield and adaptability identification, may be entered for regional trials of cultivars directly or as good parents for stripe rust resistance breeding.