In the present study, diatom samples were collected from the tropical river systems in Kerala, India. The investigation was done for a period of two years from January 2017 to December 2018. Thirty four species of diatoms belonging to fifteen genera viz. Nitzschia, Navicula, Diadesmis, Achnanthidium, Synedra, Encyonema, Sellaphora, Lemnicola, Eunotia, Pinnularia, Aulacoseira, Surirella, Stauroneis, Gomphonema and Cymbella were isolated using methods viz. serial dilution, agar plating and single cell isolation. Out of the 34 species isolated and identified, 16 species could be maintained as pure cultures in f/2 medium under laboratory conditions for further analysis viz. standardization of culture conditions and nutrient analysis. Species identification was done with the help of light microscope and scanning electron microscope images. The diatom cells were subjected to acid treatment for analyzing the structure and morphology. The study was aimed at screening, isolation and identification of the diatoms from freshwater sources and to find out its potential as live feeds in the larviculture of fin fish and shell fish species of aquaculture importance and also as a source for biodiesel production.