Background: A variety of clinically important pathogens have developed multidrug resistance (MDR), which threatens global public health. This study aimed to determine the incidence, patterns, and trends of MDR of gram-negative bacterial isolates in clinical specimens in the Tamale Teaching Hospital, Ghana. Methods: This retrospective study analyzed gram-negative bacterial isolates and antimicrobial susceptibility test (AST) results of patients who visited the Tamale Teaching Hospital laboratory between 2017 and 2019. Results: A total of 2,779 gram-negative bacterial isolates and their phenotypic AST results were analyzed. From these, 1,297 gram-negative bacteria (46.7%) were isolated from urine samples, while the rest were isolated from sputum (20.9%), wound (14.3%), and swabs (11.7%) samples, etc. Escherichia coli (23.8%) was the most common gram-negative pathogen found predominantly in the urine samples (33.2%). All gram-negative bacteria isolated between 2017 and 2019 showed high MDR. Klebsiella pneumoniae gradually increased its MDR from 84.0% in 2017, 89.5% in 2018, to 95.1% in 2019. On the other hand, the MDR rates in Pseudomonas aeruginosa were approximately 65.8%, varying from 59.5% in 2017 to 78.7% in 2019. Among tested antimicrobials, amikacin was the most effective. Resistance to amikacin in Enterobacter spp., E. coli, and K. pneumoniae in vitro were 16.2%, 11.8%, and 17.7%, respectively. Conclusion: The study has shown that the high levels of MDR in gram-negative bacteria isolated may be associated with the infections recorded at the Tamale Teaching Hospital. The major gram-negative pathogens isolated have resistance to penicillins, cephalosporins, and fluoroquinolones. Aminoglycosides can offer high antibiotic activity to overcome gramnegative bacterial resistance. Further studies will be needed to decide policy direction on infection prevention and control, and antimicrobial stewardship programs.