A novel real-time PCR (qPCR) assay with internal amplification control based on the rpoB gene was developed for the detection and quantification of Cronobacter spp. Inclusivity and exclusivity of the qPCR assay were tested on a strain collection containing 19 Cronobacter and 26 non-Cronobacter strains. All Cronobacter strains were successfully identified, whereas no cross-reactivity was observed with non-Cronobacter strains. The sensitivity of the qPCR assay for pure culture and powdered infant formula (PIF) without enrichment was 3.44 log CFU/ml(g) (2.74 x 10(3) CFU/ml(g)). When the qPCR assay was applied to artificially contaminated PIF after a 12-h enrichment step, as few as 0.03 log CFU/ml (1.06 x 10(0) CFU/ml) of C. sakazakii could be detected. The limit of detection of the qPCR assay was not reduced by the presence of 8 log CFU/ml of Salmonella Enteritidis in PIF. A total of 70 food samples were analyzed for the presence of Cronobacter spp., out of which 3 dry cereal products, 5 maternal milk, and 1 infant food formula were found as positive by qPCR. The results obtained by qPCR were consistent with those obtained by culture-based method. Results from this study demonstrate that the qPCR assay is a rapid, specific, and accurate method suitable for Cronobacter detection in foods.