The reclassification of the genus Cronobacter (previously known as Enterobacter sakazakii) was based on a polyphasic analysis that led to the description of five species. These bacteria are opportunistic pathogens that can cause neonatal meningitis and other infections in immuno-compromised individuals. Cronobacter species have been reported to show differences in sensitivity to antibiotics, heat and chemicals, as well as differences in virulence. The objective of this study was to classify Cronobacter isolates from infant formula milk, the food processing environment and fresh produce in South Africa and to evaluate the phylogenetic placement of these isolates based on the rpoA and 16S ribosomal RNA (rRNA) gene sequences. All the South African strains were identified as Cronobacter sakazakii despite the wide variety of isolation sources. No relation between the phylogenetic placement and strain origin could be determined. Strains of C. sakazakii, Cronobacter dublinensis, Cronobacter turicensis and Cronobacter muytjensii could be differentiated from each other, but it was not possible to differentiate between C. sakazakii and Cronobacter malonaticus based on the rpoA and 16S rRNA gene sequences alone. However, sequence data of these two genes can be used to differentiate between C. sakazakii and C. malonaticus when used in combination with biochemical analysis based on the utilisation of malonate.