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Background and Objectives: Antimicrobial resistance (AMR) is increasing worldwide and imposes significant life-threatening risks to several different populations, especially to those in intensive care units (ICU). The most commonly isolated organisms in ICU comprise gram-negative bacilli (GNB), and these represent a leading cause of serious infections. This study was conducted to describe the prevalence of resistance in GNB isolated from patients in adults, pediatric, and neonatal ICU in a tertiary-care hospital in Mérida, Mexico. Materials and Methods: A retrospective study was done on samples collected in Neonatal (NICU), Pediatric (PICU) and Adult (AICU) ICU of Unidad Médica de Alta Especialidad, Instituto Mexicano del Seguro Social in Mérida, México. The identification of isolates and antimicrobial susceptibility testing were performed using an automated system. Results: A total of 517 GNB strains were isolated. The most common positive culture was bronchial secretions. Pseudomonas aeruginosa was the prevalent pathogen in NICU and PICU, whereas Escherichia coli was common in the AICU. Overall, GNB exhibited a high resistance rates for Ampicillin (95.85%), Cefuroxime (84.17%), Piperacillin (82.93%), Cefotaxime (78.07%), Ceftriaxone (77.41%), Aztreonam (75.23%), Cefazolin (75.00%), and Ceftazidime (73.19%). There are significant differences in the resistance rates of GNB from different ICUs for penicillins, cephalosporins, carbapenems and fluoroquinolones drugs. Escherichia coli (multidrug-resistant [MDR] = 91.57%, highly resistant microorganisms [HRMO] = 90.36%) and Acinetobacter baumannii (MDR = 86.79%, HRMO = 83.02%) exhibited the highest percentage of MDR and HRMO profiles. The prevalence of the extended-spectrum beta-lactamases (ESBL)-producing isolates was 83.13% in E. coli, 78.84% in Klebsiella pneumoniae, and 66.67% in Proteus mirabilis, respectively. Conclusions: The high resistance rates to drugs were exhibited by our GNB isolates. Continuous surveillance and control of the use of antimicrobials are urgently needed to reduce the emergence and spreading of MDR, HRMO, and/or ESBL-producing bacilli.