Third-generation cephalosporin-resistant Enterobacterales is a major threat for newborns in neonatal intensive care units (NICUs). The route of acquisition in a non-outbreak setting should be investigated to implement adequate infection prevention measures. To identify risk factors for colonization with and to investigate the transmission pattern of third-generation cephalosporin-resistant Enterobacterales in a NICU setting. This monocentric observational cohort study in a tertiary NICU in Heidelberg, Germany, enrolled all hospitalized neonates screened for cephalosporin-resistant Enterobacterales. Data were collected from 1 January 2018 to 31 December 2021. Weekly screening by rectal swabs for colonization with third-generation cephalosporin-resistant Enterobacterales was performed for all newborns until discharge. Whole-genome sequencing was performed for molecular characterization and transmission analysis. In total, 1,287 newborns were enrolled. The median length of stay was 20 (range 1-250) days. Eighy-eight infants (6.8%) were colonized with third-generation cephalosporin-resistant Enterobacterales. Low birth weight [<1500 g (adjusted odds ratio, 5.1; 95% CI 2.2-11.5; P < 0.001)] and longer hospitalization [per 30 days (adjusted odds ratio, 1.7; 95% CI 1.5-2.0; P < 0.001)] were associated with colonization or infection with drug-resistant Enterobacterales in a multivariate analysis. Enterobacter cloacae complex was the most prevalent third-generation cephalosporin-resistant Enterobacterales detected, 64.8% (59 of 91). Whole-genome sequencing, performed for the available 85 of 91 isolates, indicated 12 transmission clusters involving 37 patients. This cohort study suggests that transmissions of third-generation cephalosporin-resistant Enterobacterales in newborns occur frequently in a non-outbreak NICU setting, highlighting the importance of surveillance and preventive measures in this vulnerable patient group. IMPORTANCE Preterm newborns are prone to infections. Therefore, infection prevention should be prioritized in this vulnerable patient group. However, outbreaks involving drug-resistant bacteria, such as third-generation resistant Enterobacterales, are often reported. Our study aims to investigate transmission and risk factors for acquiring third-generation cephalosporin-resistant Enterobacterales in a non-outbreak NICU setting. Our data indicated that premature birth and low birth weight are significant risk factors for colonization/infection with third-generation cephalosporin-resistant Enterobacterales. Furthermore, we could identify putative transmission clusters by whole-genome sequencing, highlighting the importance of preemptive measures to prevent infections in this patient collective.