RAPD analysis was performed between a near-isogenic line (NIL) Yr5/6 x Avocet S carrying the resistance gene Yr5 against wheat stripe rust and its susceptible parent Avocet S, using the Yr5 gene donor parent Triticum spelta album as control. Amplified DNA fragments were separated on 4% denaturing PAGE (polyacrylamide gel electrophoresis) and displayed by silver staining. Fifty to 100 bands were detected, 5 folds more than those revealed on agarose gels. A total of 240 random primers were screened, and 23 reproducible polymorphic DNA fragments were found, out of which 6 polymorphic bands appeared to be linked to Yr5 gene. Genetic linkage was tested on 121 segregating F2 plants derived from a cross between Avocet S and Yr5/6 x Avocet S. It was showed that the polymorphic DNA fragment S1320(207) was completely linked to Yr5 gene, and S1348(363) closely linked to Yr5 gene. The results suggested that using denaturing PAGE-silver staining could increase the level of DNA polymorphisms detected in wheat and also improve the repeatability of RAPD analysis.