Polymerase chain reaction combined with restriction fragment length polymorphism (PCR-RFLP) targeting the 16S ribosomal RNA gene (16S rDNA) provides a powerful technique for bacterial classification, especially in the identification of pathogenic strains crucial for assessing potential virulence factors such as toxins in food spoilage. This simple protocol involves PCR amplification of the 16S rDNA marker gene, followed by single or pair (co-)digestion with specific restriction endonucleases (REs). The resulting DNA fragments are then separated electrophoretically on agarose gels to identify polymorphic marker bands that essentially enable the identification of a particular bacterial strain. Here, we present a standardized 16S rDNA PCR-RFLP protocol tailored for the molecular identification of the major foodborne pathogen, Bacillus cereus sensu stricto. This protocol also serves as a robust diagnostic tool for other bacterial species, enabling it to be applicable across diverse ecological systems, including soils, animal guts, plant rhizospheres, human oral environments, etc., for various applications.