Abstract: The aim of this study was to evaluate the genetic similarity and phylogenetic relationship among four Bacillus thuringiensis subspecies (new isolate and three reference strains). Random amplified polymorphic DNA (RAPD) technique was used in this study. Based on RAPD pattern obtained with total of seventeen random primers, the similarity index was calculated for each primer separately and the average for all primers was carried out with each comparison. Moreover, the scored band data was subjected to cluster analysis using Statistica/W5.0 Software package. Based on RAPD data obtained with all primers, the average of genetic similarities between the new isolate (66) and ACCC 10061, tt4 and i977 was 42.47; 51.10; and 45.90, respectively. The four strains are grouped into two major clusters A and B. Cluster A consisted of B. thuringiensis Berliner (ACCC 10061) strain and cluster B consisted of the three B. thuringiensis (66, tt4 and i977) strains. RAPD-PCR could be used extensively to study phylogenetic relationship among B. thuringiensis strains from different subspecies as well as for identification new isolate bacterial strains.