Optimal measures for preventing and controlling carbapenem-resistant Enterobacterales (CRE) depend on their burden. This prospective observational study investigated the prevalence and clinical course of CRE colonization in hospitalized patients at Siriraj Hospital, the largest university hospital in Thailand. Stool/rectal swab samples were collected from the patients upon admission, once weekly during hospitalization and every 1–3 months after discharge, to determine the presence of CRE in the stool. Between 2018 and 2021, a total of 528 patients were included. The prevalence of CRE colonization upon admission was 15.5%, while 28.3% of patients who tested negative for CRE on admission acquired CRE during their hospitalization. CRE colonization upon admission was usually associated with prior healthcare exposure. Among CRE-colonized patients, 4.7% developed a CRE clinical infection, with 60% mortality. No cutoff period that ensured that patients were free of CRE colonization in stool was identified, and isolation precautions should only be ceased if stool tests are negative for CRE. In conclusion, the prevalence of CRE colonization among hospitalized patients at Siriraj Hospital is high. CRE-colonized patients are at risk of developing subsequent CRE infection. To prevent CRE transmission within the hospital, patients at high risk of colonization should undergo CRE screening upon admission.