Cronobacter is a genus with emerging pathogens that has been associated with life threatening diseases in neonates, infants and immunocompromised adults. Three Cronobacter species were isolated from powdered infant formula retailed in Nigeria. Different methods of phenotypic and genotypic characterization were carried out. All the isolates were identified biochemically by Microscan identification analysis as Enterobacter sakazakii (98.87%). The Vitek MALDI-TOF system identified the isolates as Cronobacter sakazakii. 16S rRNA sequencing identified the isolates as C. sakazakii. In contrast the use of species-specific PCR assays targeting rpoB, and cgcA, helped to identify two of the three strains as C. sakazakii and the last strain was identified as C. malonaticus. Multi locus sequence typing (MLST) analysis was used to identify each strain’s sequence type and the results identified three new sequence types: 303, 304 and 296. C. sakazakii BAA 894 served as a positive control for all the experiments. Biochemical methods and commercial identification systems are not sensitive enough to identify Cronobacter strains to the species level. Molecular methods are needed to confirm the species identity of strains.