Cronobacter spp. (formerly known as Enterobacter sakazakii) are foodborne pathogens that cause rare but life-threatening diseases in neonates and infants through consumption of contaminated powdered infant formula. This study was conducted to investigate the occurrence of Cronobacter spp. in powdered formula in China and to further characterize Cronobacter isolates. Isolates were identified to the species level based on the fusA gene sequence, and strains of C. sakazakii were further subtyped by applying the polymerase chain reaction (PCR)-based serotyping method. A total of 23 strains of Cronobacter spp. isolated from 530 powdered formula samples were identified using conventional biochemical methods and duplex PCR. Cronobacter spp. were detected in 6.25%, 1.82%, 3.64%, 5.45%, and 2.50% of the general formula, infant formula (age <6 months), follow-up formula (6-12 months of age), growing-up formula (1-3 years of age), and children's formula (3-6 years of age), respectively. The individual species were identified as C. sakazakii (22 isolates) and C. malonaticus (1 isolate). Among 22 C. sakazakii isolates, representatives of all but two O-antigen serotypes (serotypes O5 and O6) were recognized.