A multi locus sequence analysis (MLSA approach) was studied on the Bacillus genus, or the Bacillus subtilis species complex for specific, including 08 strains from four species (B. subtilis, B. pumilus, B. licheniformis and B. amyloliquefaciens) were provided by Biotechnology Center of Ho Chi Minh City. The research was based on sequences of 16S rRNA genes, the concatenation of five protein-coding housekeeping genes: glpF, pta, purH, pycA, and rpoD. After PCR amplification and sequencing the phylogenetic tree of 16S rRNA sequences, concatenate sequences (as well as the phylogenetic tree of each housekeeping gene) are constructed for comparison and discussion. The aim of this study is reach for better resolution and differentiation of strains and species within the B. subtilis species and to determine whether MLSA scheme show advantages in 16S rRNA gene-based studies.