Abstract Cronobacter spp. are opportunistic foodborne pathogens associated with infections in neonates and infants, particularly those that are premature or immunocompromised. Outbreaks of Cronobacter infections in infants have been epidemiologically linked to the consumption of contaminated powdered infant formulae. Since the designation of Enterobacter sakazakii (now Cronobacter spp.) as a unique species in 1980, a number of methods have been proposed for its detection and identification. The aim of the present study was to evaluate and compare different tools used for the identification of presumptive Cronobacter isolates. The bioMérieux API 20E biochemical bacterial identification kit (Omnimed (Pty), Randburg, Gauteng, South Africa) was shown not to be a reliable identification tool for the Cronobacter strains examined in the current study, since it gave false-positive results. The API 50CHE biochemical kit was shown to be more reliable giving similar results to the polymerase chain reaction (PCR) detection and DNA sequence data. The primer pair Esakf/Esakr proved to be the most reliable PCR identification tool.Additional differentiating traits and antibiotic patterns were demonstrated for Cronobacter species in the current study.