The incidence of foodborne diseases has increased over the years and has become a major global health problem. Foodborne pathogens are present in many foods, and detection of foodborne pathogens is important to ensure safe food and prevent food poisoning. Amplified fragment length polymorphism (AFLP) provides an effective method for disease genotyping, often when information about the disease genome is insufficient. In this method, restriction enzymes cut DNA, and adapters are attached to the ends of DNA fragments. AFLP analysis was developed by Marc Zabeau and detailed by Vos et al. (1995). The AFLP method was developed by combining RFLP and PCR techniques for DNA fingerprint detection. Genomic DNA is digested with restriction enzymes, and restriction fragments are preselected and selectively amplified with different primer combinations. Gram-positive pathogens are a family of toxins, the most abundant of which are the emetic and diarrheal enterotoxins of Bacillus cereus, the neurotoxins of Clostridium botulinum, the enterotoxins of Clostridium perfringens, and the enterotoxins produced by Staphylococcus aureus and other staphylococci. These toxins are the most important cause of many diseases in food and are the main causes of food contamination. Bacteria are considered a major cause of food poisoning. These are Bacillus cereus, Clostridium botulinum, and Staphylococcus aureus, all of which can cause disease by producing toxins in food. This chapter summarizes the detection of foodborne Gram-positive bacterial toxins by amplified fragment length polymorphism.