Cereals are the foremost necessary foods for growing population of human. Wheat (Triticum aestivum) is the first important and strategic cereal crop for the majority of world’s populations. It is the most necessary staple food. Although rice (oryzae sativa) has the second place because of planted area but it serves as the most important food source for Asian countries. Wheat and rice production may be severely restricted by biotic and abiotic constraints. Disease is the major biotic stress in several regions.Comparative sequence analysis is a powerful tool to study homologous gene families, define conserved gene functions between orthologs, and identify lineage- and species-specific genes. Most annotations of newly sequenced genomes are based on similarity with sequences for which functional information is available. Apart from conserved sequences, inter-species differences provide important clues about evolutionary history and species-specific adaptations. In our study, two RNA-sequencing data sets of resistant variety of wheat (Triticum aestivum L.,) after infection with leaf rust fungus, Puccinia triticina and resistant variety of rice (Oryza sativa L.,) after infection with blast fungus, Magnaporthe oryzae were compared. 31768 up-regulated genes in wheat and 3902 up-regulated genes in rice were filtered according to fold change more than 3 and removing variants, 250 upregulated genes of wheat and rice were aligned and phylogenetic tree was generated. The result of phylogenetic tree showed close relationship between ten aligned gene pairs of wheat and rice. Two pairs of aligned gene pairs were selected randomly, super family of these pairs were obtained, the result showed that each aligned pair of proteins shared the same protein family and the same annotation and all pairs participate in plant defense pathways. Then, the gene expression of the two pairs were validated by Real-time PCR after infecting wheat with Puccinia triticina and rice with Magnaporthe grisea. Each aligned pair of the two pairs shared the same manner of expression with few exceptions in rice.