A large number of Bacillus thuringiensis (Bt) isolates separated from different ecological regions of Pakistan were characterized for crystal protein gene composition and pesticidal activity against two lepidopteran rice insect pests, the yellow stem borer (Scripophaga incertulas) and the rice leaf folders (Cnaphalocrocis medinalis). A representative seventeen isolates were and the rice leaf folders (Cnaphalocrocis medinalis). A representative seventeen isolates were selected on the basis of initial screening and further characterization of pesticidal activity was performed according to following criteria; colony and parasporal inclusion morphology, SDS-PAGE, western blot analysis and comparative biotoxicity assays to determine ${LC}_{50}$ values. All isolates produced parasporal inclusion bodies and spores in their cells. Immunoblotting results showed that Pakistanian isolates synthesized entomocidal proteins belonging to Cry1A and Cry2A toxin groups. the biological activity of local isolates demonstrated a wide range of ${LC}_{50}$ values against both target insects pests. The most potet isolates, INS 1.13, INS 2.25 and NW 4.1 against S.incertulas showed ${LC}_{50}$ values of 29.83, 30.37 and 24.77 ng/ml of toxin, respectively. The ${LC}_{50}$ values of 57.37 and 73.09 ng/ml of toxin were exhibited by local isolates, INS 2.25 and RL 4.8 against C. medinalis, respectively.