Carbapenem-resistant Klebsiella pneumoniae (CRKP) pose an emerging clinical threat. We investigated its introduction and transmission in a new hospital, evaluating the effect of whole-genome sequencing (WGS) as an infection control measure. Based on WGS of identified K. pneumoniae (Kpn) strains, a prospective molecular epidemiological study of nosocomial transmission of CRKP in a newly established Chinese hospital was conducted. Between September 2018 and August 2020, 206 Kpn strains were isolated, including 180 CRKP, from 152 patients. The first imported and nosocomial transmission cases were recorded in December 2018 and April 2019, respectively. Overall, 22 nosocomial transmission clusters involving 85 patients were identified, among which 5 were large-size clusters comprising 5-18 patients. Index cases of the large-size clusters were more likely associated with lower Glasgow Coma Scale scores than those of small-size clusters. Furthermore, results of multivariable logistic regression indicated that Kpn tended to transmit more among patients in the ICU [adjusted odds ratio (aOR)=4.96, 95% confidence interval (CI) 1.97-13.47] and those infected with a ST11 strain (aOR=8.04, 95% CI 2.51-29.53) or tetracycline-resistant strains (aOR=17.63, 95% CI 6.32-57.32). However, transmission was less likely in strains bearing the rmpA gene (aOR=0.12, 95% CI 0.03-0.37). The rate of nosocomial CRKP cases decreased by 2.25 with the intervention of WGS-based infection control. Kpn transmission in the newly established hospital originated from several imported cases. Rates of nosocomial CRKP infection were reduced considerably through precise infection control measures.