The efficacy of the restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified rrs (16S ribosomal ribonucleic acid [rRNA] gene) for the identification of Bacillus spp. isolated from freshwater pond ecosystems was evaluated. Results showed that no restriction site differences occur between rrs operons within the bacteria belonging to the same species and as a result members of the same species yield very similar RFLP patterns at 100% similarity level. Cluster analysis of PCR-RFLP patterns grouped all the isolates into three clusters; each cluster represented a single species except one for which no placement was obtained. Phenotypic characteristics of the isolates were found to be well concordance with the results of PCR-RFLP. Our findings suggested that PCR- RFLP is suitable tool for discrimination and identification of endoglucanase positive Bacillus spp. isolated from freshwater ecosystems.