Objective: To perform comparative diagnostic study with rotavirus infected stool samples for efficacy ofindigenously prepared molecular transport medium (IMTM).Study Design: It was an experimental study.Place and Duration of Study: The study was conducted at Department of Microbiology of University of Karachi,Karachi Pakistan from June to July 2021.Materials and Methods: The current study was conducted based on hospital based surveillance of rotavirusgastroenteritis among children <5 years of age, stool samples were collected in commercially available viraltransport medium. Samples were known rotavirus positive /negative through PREMIER Rota clone Enzyme-Linked Immunosorbent Assay (ELISA) kit diagnostic test. Total 64 stool samples were selected to evaluate theefficacy of indigenous molecular transport medium (IMTM). Stool samples were spiked in IMTM andcommercial available viral transport medium (CVTM). RNA extraction and Real-time PCR were performed byQIAmp and in house assay respectively as per manufacturer' instructions. Similar method of RNA extractionand Real-Time PCR for both type of spiked samples was adopted.Results: Comparative analysis of Cycle threshold (Ct) values of paired samples (IMTM vs CVTM) was performed.Real-Time (RT-PCR) for rotavirus positive and negative results were consistent with each pair (p<0.0001).Conclusion: The indigenously formulated IMTM found capable for stool sample collection, transportation andprocessing for rotavirus detection through Real-time PCR.