Aeromonads possess an array of virulence factors and are causative agents of a number of human infections. Among them, genes of one cytotoxic (Act) and two cytotonic (Alt, Ast) enterotoxins are implicated in a human diarrheal disease. A rapid, specific, simultaneous detection of these enterotoxin genes in suspected food poisoning samples is not yet reported. Hence, a multiplex PCR assay was designed to amplify the cytotoxic (act), heat-labile cytotonic (alt), and heat-stable cytotonic (ast) enterotoxin genes of aeromonads. The PCR assay was tested with 133 Aeromonas spp. isolated from suspect food poisoning samples and retail samples of poultry and fish from wet markets in and around Taipei, Northern Taiwan. The Aeromonas spp. isolates were divided into six genotypes based on absence or presence of one or more enterotoxin genes. Of these 133 isolates, Aeromonas caviae (52.5%) and Aeromonas hydrophila (43.4%) were the most frequently isolated species from food poisoning samples and retail samples, respectively. Among the species, A. hydrophila had a significantly higher proportion for harboring three enterotoxin genes than had the others, whereas Aeromonas encheleia, considered a nonpathogen, was found harboring three enterotoxin genes. The multiplex PCR assays are rapid and specific, and provide a useful tool for the detection and genotyping of enterotoxin genes of aeromonads.