The aim of the present study is to develop and characterize single nucleotide polymorphism (SNPs) markers from Expressed Sequence Tags (EST) linked to rust resistance genes in wheat. The use of genetic resistance is the most profitable and environmentally friendly strategy for farmers to control wheat rusts in both the developing and the developed world. Looking for resistance genes and designing primer are an important goal in studying the mechanism of crop culture and the pathogen. A bioinformatics strategy was used to identify SNPs within large ESTr esources. An integrated SNP discovery pipeline was developed, using the Perl script and SNPfinder application that identify SNPs from assembled EST sequences. Users can rapidly identify polymorphic sequences of interest through MegaBLAST sequence comparisons. A total of 5,666 ESTs for rust resistance genes were downloaded from GenBank. Additional comparative analyses as well as searches for protein domains, multiple alignments and construction of phylogenetic trees were performed to confirm the presence of genes of interest. Five hundred and twenty seven SNPs were identified for rust resistance genes,I ncluding 449 in bread wheat (321 linked to leaf rust resistance genes and 128 linked to stem rust resistance genes). T/G is the most common SNP structure type for leaf rust, and G/A for stripe rust. In durum wheat, 78 SNPs linked to leaf rust resistance were identified; the most frequent structure type was T/G. Sixty three primers pairs from leaf and stripe rust sequences of the Triticum genus, were identified and analyzed for in-silico design of PCR primers. The identified SNPs linked with useful biological information will be valuable resources for functional genomics and molecular wheat breeding applications. 020357