Carbapenems are group of extended-spectrum β-lactam antimicrobials frequently used for treating multidrug-resistant Gram-negative bacilli (GNB) infections. This study aimed at detecting and characterizing carbapenem resistance (CR) genes among GNB isolated from patients treated in neonatal intensive care unit (NICU) of Mansoura University Children's Hospital (MUCH), Egypt. It is a prospective study conducted from 2015 to 2016. A total of 158 GNB isolates were examined for CR both phenotypically and genotypically. Among 158 Gram negative isolates, there were 58 (36.7%) CR strains. Extended-spectrum β-lactamase (ESBL) production was confirmed in all 58 (100%) isolates. Carbapenemase production was detected in 52 (89.5%) strains while metallo beta-lactamase (MBL) production was found in 33 (56.9%) strains. Molecular characterization of CR strains revealed that 57 (98.3%) isolates were positive for carbapenemase encoding genes. KPC gene was the most frequent detected gene (34/58). VIM, IPM, OXA and NDM genes were also detected in 15, 13, 9 and 1 isolate, respectively. Only one isolate was negative for all encoding resistance genes despite positive for ESBL phenotype. Infection with CR strains has been increasing in clinical settings which limit the use of carbapenems.