Background: Recently, the spread of NDM-5-producing Escherichia coli has become a severe challenge in clinical therapy, which necessitates reliable detection and surveillance methods. However, limited information is available regarding the prevalence and dissemination of the blaNDM-5 gene in Escherichia coli in China. Therefore, we investigated the dissemination of the blaNDM-5 gene in carbapenem-resistant Escherichia coli isolates from different regions in China.Methods: A total of 1,180 carbapenem-resistant enterobacteriaceae strains were obtained from patients admitted to the 20 sentinel hospitals in eight cities. Strains positive for blaNDM-5 were detected using the Vitek 2 compact system, 16S rRNA gene sequencing, PCR, the S1-pulsed-field gel electrophoresis assay, and Southern blot hybridization. The horizontal-transfer capability of the blaNDM gene was assessed by filter mating with a standard E. coli J53 azide-resistant strain as the recipient. Genotyping, susceptibility testing, and whole genome sequencing were performed. Results: Seven strains of blaNDM-5-positive E.coli was detected in 1180 clinical strains from different regions in China. The blaNDM-5-carrying strains showed resistance to multiple tested antibiotics and belonged to two widespread sequence types, ST167 and ST405. Antimicrobial resistance genes including blaCTX-M, blaOXA, blaCMY, and two novel blaTEM variants (blaTEM-230 and blaTEM-231) were also identified. Southern blotting located the blaNDM-5 gene on 46-kb IncX3 plasmids in all isolates, which showed only two single nucleotide differences between EJN003 and the other strains. Conclusions: This study further confirms the increasing occurrence of blaNDM-5-carrying IncX3 plasmids and the dissemination of carbapenem resistance in E. coli isolates via the plasmid from different parts in China, which warrants stringent surveillance and control measures.