ObjectivesTo describe pathogens distribution and their antimicrobial resistance (AMR) of neonatal bloodstream infections (BSIs) at 25 NICUs in eastern China, 2016-2019.Study designWe conducted a multicentre retrospective descriptive analysis of all positive blood cultures collected at 25 NICUs of tertiary hospitals between January 1, 2017, and December 31, 2019. ResultsFrom 1 January 2017 to 31 December 2019, a total of 1092 pathogenic bacteria isolates were met inclusion criteria among 66516 specimens. 349 (32%) isolates were responsible for early-onset sepsis (EOS), 702 (64.2%) hospital-acquired late-onset sepsis (HALOS), and 41(3.8%) community-acquired late-onset sepsis (CALOS). The majority isolates causing EOS (198/349, 56.7%) and HALOS (437/702, 62.2%) were gram-negative bacteria; The most frequent pathogens causing EOS were Escherichia coli (95/349, 27.2) and group B streptococcus (GBS; 51/349, 14.6%). E coli were primarily identified among preterm infants (58/95, 61.1%) with GBS primarily among term infants (43/51, 84.3%). GBS and Staphylococcus aureus were responsible for 46.3% (19/36) and 41.5% (17/36) of episodes of CALOS. Isolates causing HALOS were more often resistant than isolates causing EOS and CALOS. K. pneumoniae (196/702, 27.9%) as the most common isolates responsible for HALOS were more likely to be resistant to third-generation cephalosporins (124/196, 63.3%), gentamicin (41/196, 20.9%) and carbapenem (26/196, 13.3%). Conclusions Escherichia coli, K. pneumonia and GBS were the most common pathogens in EOS, HALOS and CALOS, respectively. High AMR in K. pneumoniae causing HALOS warrants continued study and devise measures to prevent it in low-income and middle-income countries.