Background: The widespread dissemination of carbapenem-resistant Gram-negative bacteria poses a significant threat to global public health., Purpose: This study aimed to investigate the prevalence of carbapenem resistance in Gram-negative bacteria isolated from patients at the Children's Medical Center Hospital, Tehran, Iran, to understand the molecular mechanisms underlying this resistance., Methods: During the period spanning from June 2019 to June 2020, 777 Gram-negative bacterial strains were isolated. Antibiotic susceptibility testing was performed according to Clinical and Laboratory Standards Institute. Polymerase chain reaction was used to detect carbapenem resistance genes including bla OXA23, bla OXA24, bla OXA48, bla OXA51, bla OXA58, bla OXA143, bla KPC, bla IMP, bla VIM, and bla NDM., Results: Among the total bacterial isolates, 141 (18.1%) exhibited carbapenem resistance. Escherichia coli was the most prevalent (57.4%), followed by Klebsiella pneumoniae (11.3%) and Acinetobacter baumannii (10.6%). Other notable contributors included Enterobacter spp. (5.7%), Salmonella spp. (3.5%), and Stenotrophomonas maltophilia (2.8%). Citrobacter spp., Proteus mirabilis, and Pseudomonas aeruginosa contributed to the distributions of two, one, and three isolates, respectively. Notably, bla OXA48 showed the highest prevalence (33%), followed by bla OXA143 and bla OXA58 (27% and 24%, respectively). In addition, bla OXA24 was present in 11% of the total isolates, bla OXA23 in 10%, and bla NDM in 10%, whereas bla KPC, bla VIM, and bla IMP were not detected., Conclusion: Our study highlights the prevalence of carbapenemase-producing Gram-negative isolates among pediatric patients. Notable resistance patterns, especially in K. pneumoniae and E. coli, underline the urgent need for proactive interventions, including appropriate antibiotic prescription practices and strengthening of antibiotic stewardship programs.