The spread of multi-drug resistant microbial strains and reduced range of drugs available led to an enquiry for therapeutic alternatives. Medicinal plants and peptides isolated from them are being used for their antimicrobial properties. Plant defensins are short peptides that show resistance against a variety of human and plant pathogens with a number of other bioactivities and characteristics. Antimicrobial peptides have also been isolated from Solanaceae family. We therefore focused our research towards isolation of defensin-like genes from two selected plants of Solanaceae family which were Solanum tuberosum and Withania somnifera. Plants genomic DNA were isolated by using CTAB extraction method. The presence of DEFL genes were confirmed by PCR with specifically designed primers in both plants and results were established by using agarose gel electrophoresis. The purified desired PCR product bands of 1100 bp from Withania somnifera & 1000 bp Solanum tuberosum were sent for sequencing for further studies. This research work can further help to comprehend the genetic & functional diversity of the defensin like peptide, their distinctiveness, regulation, and better research based to understand the topic.