Objective To analyze the resistance mechanisms and molecular epidemiological characteristics of carbapenem-resistant Enterobacterales (CRE) from urine, and to provide scientific basis for the prevention, control, and treatment of CRE-associated urinary tract infection. Methods A total of 97 CRE strains isolated from urine specimens from the First Affiliated Hospital of Nanchang University from 2021 to 2024 were collected, and antimicrobial susceptibility testing was performed with VITEK 2 Compact automatic microbial identification and antimicrobial susceptibility analysis system. The resistance genes, plasmid types, and molecular epidemiological characteristics of strains were analyzed by whole-genome sequencing technique. Results Among the sequenced 97 CRE strains, Klebsiella pneumoniae (n=75, 77.32%) was predominant, followed by Escherichia coli (n=9, 9.28%) and Enterobacter cloacae (n=9, 9.28%). Most strains were mainly from the department of rehabilitation medicine (n=37, 38.15%). Antimicrobial susceptibility testing revealed that the resistance rate of CRE to most commonly used antimicrobial agents exceeded 90%; 92.78% (90/97) of CRE carried carbapenemase genes, with only two genotypes (bla_KPC and bla_NDM) being detected, primarily the bla_KPC-2 and bla_NDM-5 subtypes. The plasmid types carried by CRE exhibited high diversity, with the number of plasmid replicons ranging from 1 to 11. 94.85% of the strains harbored three or more plasmid replicons, with IncFII, IncR, and IncHI1B being the most prevalent. Multilocus sequence typing (MLST) results showed that ST11-KL64 and ST11-KL25 were the predominant clones of carbapenem-resistant Klebsiella pneumoniae (CRKP), while carbapenem-resistant Escherichia coli (CREC) and carbapenem-resistant Enterobacter cloacae (CRECL) exhibited significant clonal diversity. Conclusion CRE resis-tance in urinary tract infection in this region is severe, with CRE mainly carrying bla_KPC-2 and bla_NDM-5 genes, with highly diverse plasmid types. In addition, the ST11-KL64 and ST11-KL25 types are the dominant clones of CRKP, while other CREs exhibit significant clonal diversity, monitoring and prevention should be enhanced.