This investigation was designed to induce a foreign genomic fragments of Bacillus thuringiensis (which is considered as important soil bacteria due to its ability to produce a lethal protoxin) to calli of three sugarcane varieties namely, C0 413, PH 8013 and GT 54-9. Thus, protoplast fusion technique was performed using Agrobacterium tumefaciens and Bacillus thuringiensis to produce a fusionts bacterium. Then, calli of different varieties under study were infected with parental and fusionts bacteria. To detect the transformants for sugarcane calli, SDS-PAGE technique was used. Consequently, successful expression of target gene was detected through produce a final protoxin (target band with 116 KDa) which characterize Bacillus thuringiensis as a bio insecticidal in all three varieties which were infected with delta endotoxin fusionts. Finally, polymorphism of DNA fragments, produced using arbitrary primers was study via RAPD technique and genetic similarity was recorded. In order to detect the differences between natural and transformant varieties, the  percentages of polymorphism were recoded and found 18, 21, 5 and 45 % for first, second, third and fourth primers respectively.