The Bacillus subtilis group (Bs group), with Bacillus subtilis as its core species, holds significant research and economic value in various fields, including science, industrial production, food, and pharmaceuticals. However, most studies have been confined to comparative genomics analyses and exploration within individual genomes at the level of species, with few conducted within groups across different species. This study focused on Bacillus subtilis, the model of Gram-positive bacteria, and 14 other species with significant research value, employing comparative pangenomics as well as population enrichment analysis to ascertain the functional enrichment and diversity. Through the quantification of pangenome openness, this work revealed the underlying biological drivers and significant correlation between pangenome openness and various factors, including the distribution of toxin–antitoxin- and integrase-related genes, as well as the number of endonucleases, recombinases, repair system-related genes, prophages, integrases, and transfer mobile elements. Furthermore, the functional enrichment results indicated the potential for secondary metabolite, probiotic, and antibiotic exploration in Bacillus licheniformis, Bacillus paralicheniformis, and Bacillus spizizenii, respectively. In general, this work systematically exposed the quantification of pangenome openness, biological drivers, the pivotal role of genomic instability factors, and mobile elements, providing targeted exploration guidance for the Bs group.