Aside from applications in the production of commercial enzymes and metabolites, Bacillus amyloliquefaciens is also an important group of plant growth-promoting rhizobacteria that supports plant growth and suppresses phytopathogens. A host-genotype-independent counter-selectable marker would enable rapid genetic manipulation and metabolic engineering, accelerating the study of B. amyloliquefaciens and its development as both a microbial cell factory and plant growth-promoting rhizobacteria. Here, a host-genotype-independent counter-selectable marker pheS (*) was constructed through a point mutation of the gene pheS, which encodes the alpha-subunit of phenylalanyl-tRNA synthetase in Bacillus subtilis strain 168. In the presence of 5 mM p-chloro-phenylalanine, 100 % of B. amyloliquefaciens strain SQR9 cells carrying pheS (*) were killed, whereas the wild-type strain SQR9 showed resistance to p-chloro-phenylalanine. A simple pheS (*) and overlap-PCR-based strategy was developed to create the marker-free deletion of the amyE gene as well as a 37-kb bmy cluster in B. amyloliquefaciens SQR9. The effectiveness of pheS (*) as a counter-selectable marker in B. amyloliquefaciens was further confirmed through the deletion of amyE genes in strains B. amyloliquefaciens FZB42 and NJN-6. In addition, the potential use of pheS (*) in other Bacillus species was preliminarily assessed. The expression of PheS(*) in B. subtilis strain 168 and B. cereus strain ATCC 14579 caused pronounced sensitivity of both hosts to p-chloro-phenylalanine, indicating that pheS (*) could be used as a counter-selectable marker (CSM) in these strains.