Cellulose is one of the most abundant and renewable biomass products used for the production of bioethanol. Cellulose can be efficiently hydrolyzed by Bacillus subtilis VS15, a strain isolate obtained from decomposing logs. A genome shuffling approach was implemented to improve the cellulase activity of Bacillus subtilis VS15. Mutant strains were created using ethyl methyl sulfonate (EMS), N-Methyl-N′ nitro-N-nitrosoguanidine (NTG), and ultraviolet light (UV) followed by recursive protoplast fusion. After two rounds of shuffling, the mutants Gb2, Gc8, and Gd7 were produced that had an increase in cellulase activity of 128%, 148%, and 167%, respectively, in comparison to the wild type VS15. The genetic diversity of the shuffled strain Gd7 and wild type VS15 was compared at whole genome level. Genomic-level comparisons identified a set of eight genes, consisting of cellulase and regulatory genes, of interest for further analyses. Various genes were identified with insertions and deletions that may be involved in improved celluase production in Gd7. Strain Gd7 maintained the capability of hydrolyzing wheatbran to glucose and converting glucose to ethanol by fermentation with Saccharomyces cerevisiae of the wild type VS17. This ability was further confirmed by the acidified potassium dichromate (K2Cr2O7) method.