Aims: To identify Bacillus species and related genera by fingerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classification of Bacillus species and related genera. Methods and Results: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from five newly created Bacillus-related genera, and five species from five phenotypically related genera were tested. A,total of 77 distinct 16S :rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene fingerprinting and the 23S and 5S rRNA genes fingerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. Conclusions: The Bacillus species along with the species from related genera were identified successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. Significance and Impact of the Study: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, abut does not duplicate, phylogenies based on 16S rRNA gene sequences.