Bacillus thuringiensis Berliner is a gram-positive, spore forming bacterium that produces proteinaceous crystal containing insecticidal toxins. The pathogenicity of 20 B. thuringiensis isolates, collected from the Western Ghats of Kerala, were evaluated. The tobacco caterpillar, Spodoptera litura Fb. was used as the test insect. Three isolates were found to be toxic to S. litura during preliminary screening. A standardized bioassay carried out with five different concentrations of all the selected isolates and the reference standard HD-1, indicated the equal effectiveness of the native isolates with the standard strain. The lowest LC50 was obtained in KAU-51 and highest in KAU-11. The lowest LT50 was recorded in HD-1.The molecular characterization of the selected isolates was performed with RAPD-PCR technique. Ten primers, which produced more than five bands were selected for RAPD analysis. The RAPD data was used to generate a similarity matrix using the NTSyS programme. The results showed that high variability exists between the selected isolates.