Objective To analyze the clinical characteristics and risk factors of hospitalized neonates with carba-penem-resistant Enterobacterales (CRE) colonization/infection. Methods Anal swab active screening and identification were conducted on neonates admitted to the neonatology departments of 5 hospitals from January to December 2021 at admission and once a week in a prospective cohort study. Information on distribution of Enterobacterales infection sites in neonates were simultaneously collected. Based on anal swab identification, neonates from whom CRE were isolated were in CRE group and whom with carbapenem-sensitive Enterobacterales (CSE) were in CSE group. The species distribution and resistance spectrum characteristics of CRE strains were analyzed. Independent risk factors for neonatal CRE colonization/infection were identified through multivariate analysis. Results A total of 483 neonates were included for active screening and identification through anal swabs upon admission and once a week. CRE were isolated from 54 cases and CSE from 98 cases. In CRE group, 22 cases (40.74%) were colonized and 32 (59.26%) were infected. In CSE group, 42 cases (42.86%) were colonized and 56 cases (57.14%) were infected. The major infection site of neonates infected with CRE and CSE was lung, accounting for 84.38% and 60.71%, respectively. CRE and CSE strains detected through anal swabs were mainly Klebsiella pneumoniae, accounting for 46.30% and 56.12%, respectively. Antimicrobial susceptibility testing results of CRE showed that Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli exhibited fully resistance (100%) or high resistance (>90%) to most β-lactam and carbapenem antibiotics, while were relatively sensitive to aminoglycosides (such as amikacin, gentamicin, polymyxin B, tigecycline), peptides, and tetracyclines. Multivariate logistic regre-ssion analysis suggested that 1-minute Apgar score≤7, sputum aspiration, and parenteral nutrition were independent risk factors for neonatal CRE colonization/infection (all P<0.05). Conclusion The incidence of CRE colonization/infection in hospitalized neonates is relatively high, and the detected strains are mainly Klebsiella pneumoniae. It is recommended to implement preventive and control measures such as screening at admission and regularly, contact isolation, standardized procedures, as well as antimicrobial management for neonates with post-birth 1-minute Apgar score≤7 and those receiving sputum aspiration or parenteral nutrition in neonatology department.