BackgroundEscherichia coliis one of the most common pathogens causing neonatal infections. Recently, the incidence and drug resistance ofE. colihave increased, posing a major threat to neonatal health. The aim of this study was to describe and analyze the antibiotic resistance and multilocus sequence typing (MLST) characteristics ofE. coliderived from infants admitted to neonatal intensive care units (NICUs) across China.MethodsIn this study, 370 strains ofE. colifrom neonates were collected.E. coliisolated from these specimens were subjected to antimicrobial susceptibility testing (by broth microdilution method) and MLST.ResultsThe overall resistance rate was 82.68%, with the highest rate of methicillin/sulfamethoxazole (55.68%) followed by cefotaxime (46.22%). Multiple resistance rate was 36.74%, 132 strains (35.68%) had extended-spectrum β-lactamase (ESBL) phenotype and 5 strains (1.35%) had insensitivity to the tested carbapenem antibiotics. The resistance ofE. coliisolated from different pathogenicity and different sites of infections varied, strains derived from sputum were significantly more resistant to β-lactams and tetracyclines. Currently, the prevalence spectrum in NICUs was dominated by ST1193, ST95, ST73, ST69 and ST131 across China. And the multidrug resistance of ST410 was the most severe. ST410 had the highest resistance rate to cefotaxime (86.67%), and its most common multidrug resistance pattern was β-lactams + aminoglycosides + quinolones + tetracyclines + sulfonamides.ConclusionsSubstantial proportions of neonatalE. coliisolates were severely resistant to commonly administered antibiotics. MLST results can suggest the prevalent characteristics of antibiotic resistance inE. coliwith different ST types.