Pleurotus sp is an edible mushroom, which contains essential nutrients and medicinal properties for living thing. Molecular characterization and genome differentiation of pleurotus sp. studied using gel electrophoresis, RAPD and PCR techniques. Separated DNA was amplified using internal transcribed spacer primers. 5.8s rRNA sequence submitted to Genbank for comparison and to obtain accession number. Differentiation and identification of Pleurotus sp found by similarity searches (Accession number: JQ740170 and JQ740171). Comparative studies have shown these two Pleurotus sp (spp1 and ssp2) are closely related to species, 95% - 100% identical, Pleurotus Citrinopileatus, Pleurotus populinus, Pleurotus ostreatus, Pleurotus abieticola, Pleurotus pulmonarius, Pleurotus eryngii and Pleurotus sapidus. These Pleurotus sp have been studied for exhibiting pharmacological potential activities such as antioxidant, antitumor, immunomodulatory activity and hypocholesterolemic through sequence similarity, nucleotide composition, evolutionary studies, secondary structure prediction and conserved region. The study extended to identify the significance of species diversity and fungal samples among various applications. Pleurotus (sp) sample was subjected to identification of bioactive compounds by using GC-MS. In Pleurotus spp1: Pyridine- 3-carboxamide, 4-dimethylamino-N-(2, 4-difluorophenyl), Piperidin-4-carboxylic acid, Aspidofractinine-3-methanol, (2à, 3á, 5à), Indolizine, and 2-(4-methylphenyl)-. Pleurotus spp2: Imidazolidine, 1, 3-dinitro, Phenol, 2-methyl-4-(1, 1, 3, 3-tetramethylbutyl), and Squalene. Enhancement of more entries as well as sequence comparison and virtual screening might help researchers to acknowledge real world problems between cultivating nutritional, medicinal and poisonous mushroom.