Members of the genus Cronobacter were once classified as Enterobacter sakazakii. These bacteria are opportunistic pathogens that are associated with necrotizing enteritis, sepsis and meningitis in neonatal or low-birth-weight infants and can result in death, slowed development, or extensive neurological disorders. In adults, they have been documented as a cause of bacteremia, osteomyelitis, and vaginitis. Previously, E. sakazakii was found in the midgut of stable flies. Research by Nangoh et al. determined that Cronobacter spp. (previously identified as E. sakazakii) are present in bison and bovine feces. In addition to the bison and bovine fecal isolates of Cronobacter spp. found by Nangoh et al., other isolates of Cronobacter spp. were analyzed phenotypically and genetically for biochemical typing and genotyping. The additional isolates include several American Type Culture Collection isolates, an isolate from a neonatal meningitis case, and multiple isolates of various origins received from Cornell University. These isolates were further categorized using four different biochemical tests. The results of these tests placed the isolates into one of the six different species or subspecies within the genus Cronobacter. For genotyping, the isolates were tested for the gene specifically responsible for the a-glucosidase activity. In addition, Pulsed-Field Gel Electrophoresis using two different enzymes, Xbal and Spel, was performed to determine possible genetic similarity of isolates from the bison and bovine feces to other isolates found in food, clinical and environmental settings. The X'baI enzyme showed two Cornell isolates, F6-049 and F6-051, had a high degree of similarity; both of these isolates were from the same clinical source. Isolates from bison and bovine feces, 52 and N72, respectively, have a high degree of dissimilarity to each other, ~ 75%. Isolate 52 showed ~ 35% dissimilarity to an isolate from a food source, and N72 showed ~45% dissimilarity to an isolate from a clinical source. The results using the Spel enzyme showed a wide diversity among the isolates. This study shows that very few of the Cronobacter spp. isolates are closely related and that there is a high level of diversity based on pulse-field gel electrophoresis and biochemical analysis.