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The phenomenon of a nonrandom distribution of mutations in a genome has been observed for many years. In fact, recent findings have indicated the presence of mutation clusters in different biological systems, including chemically treated yeast, transgenic mice, and human cancer cells. Until now, an asymmetrical distribution of mutations was only described in a single bacterial species. Here, we used ethyl methanesulfonate mutagenesis and a whole-genome sequencing approach to determine if this phenomenon is universal and not confined to Gram-negative bacteria. The Gram-positive bacterium <i>Bacillus subtilis</i> was selected for ethyl methanesulfonate treatment, followed by the next-generation sequencing of several mutagenized <i>B. subtilis</i> genomes. A nonrandom distribution of mutations was observed. This pilot study with a limited number of sequenced clones may indicate not only the universality of the phenomenon of mutation clusters but also the effectiveness of the use of a whole-genome sequencing approach in studying this phenomenon.