Objective To evaluate the colonization of carbapenem-resistant Klebsiella pneumoniae (CRKP) in neo- nates in neonatal intensive care unit (NICU), and analyze the risk actors for development of clinical infection from colonization. Methods Neonates in NICU of a hospital from January 2017 to December 2018 were selected as the research object. Since January 2017, active screening of CRKP anal swab/pharyngeal swab was carried out once a week within 48 hours after admission and during hospitalization period, monitoring on CRKP infection was performed at the same time, data was analyzed with SPSS 23.0 statistical software. Results A total of 1 230 NICU neonates were actively screened, 110 neonates were CRKP positive, CRKP colonization rate was 8.9%, 8.1% (97/1 197) of which was intestinal colonization, which was significantly higher than that (5.2%, 49/945) of the upper respiratory tract, difference was significant (P=0.008). Gestational age, low birth weight and prolonged hospital stay were risk factors for CRKP colonization (all P < 0.001). Diversilab homology monitoring found that the probability of CRKP colonization developing into clinical infection was 19.1% (21/110). Mechanical ventilation (OR, 10.177[95%CI, 2.667-38.830], P=0.013), high assessment on neonatal nutritional risk (OR, 0.251[95%CI, 0.072-0.881], P=0.031) and high score for neonatal acute physiology-Ⅱ (SNAP-Ⅱ) (OR, 8.256[95%CI, 6.072-20.881], P=0.025) were independent risk factors for the development of CRKP clinical infection in CRKP-colonized neonates. Conclusion CRKP colonization increases the probability of CRKP infection in NICU neonates during hospitalization, neonates receiving mechanical ventilation, malnutrition and high SNAP-Ⅱ score should be paid attention, so as to reduce the occurrence of CRKP clinical infection.