In some of these studies, primers were designed for the amplification of this gene, however they were not adequate for the amplification of the species of Bacillus used in this study. Accordingly, in this study a new pair of primers for the amplification of the tuf gene was designed and used for the amplification of Bacillus spp. relevant for food safety. The phylogenetic trees constructed with the tuf gene and 16S rRNA gene were compared. Based on the results obtained, the tuf gene resulted to be more discriminant for the differentiation of closely related species such as B. subtilis and B. amyloliquefaciens than other genes or that MALDI-TOF. In contrast, all gene markers showed that the discrimination between B. cereus and B. thuringiensis is quite difficult despite the variability within the tuf, rpoB and gyrB genes. In this regard, similar results were found when using MALDI-TOF for the discrimination the B. cereus group