For the majority of world populations, wheat (Triticum aestivum L.) would be the first essential and economic cereal grain crop. Pests and pathogens in both rich and developing countries are constantly threatening wheat production and sustainable development. Multiple gene pathways were recorded to share an association with fungal pathogens with wheat biological resistance. Our aim to use such tools in order to detect and classify fungal resistance genes in wheat through sequence alignment, protein domain identification and phylogenetic analysis. In addition the introduction for restriction fragment length polymorphism (RFLP) for such genes in the new primer database. Approximately 138 sequences of DNA were recovered from the wheat genome by aligning 3845 anti-fungal amino acids through tblastn tool. The NCBI blastn online tool used to detect sequences with functional genes, where 92 genes have been detected. The total number of nucleotides was 48385, where the smallest DNA sequence have 302 bp and the longest contains 977 bp with an average length of 525.9 bp per sequence. The wheat chromosomes 3D, and 4B have the highest number of sequences (9) followed by chromosomes 3B (7) and 3A(6), where wheat genomes A, B and D have 30, 35 and 27 genes, respectively. Five different amino acids motifs have been revealed among studied wheat amino acid sequences. The gene annotation tools used to infer studied amino acid gene annotation. Amino acid sequences belongs to lectin, kinase, tyrosine-protein kinase (STK), thaumatin, and cysteine-rich repeats representing 2, 9, 8, 19, 23 genes respectively, in addition to 31 hypothetical genes. The proteins chemical content have been assessed through 16 different amino acid chemical and physical characteristics.