is a major public health concern due to its role in Gram-negative bacteremia, which leads to high mortality and increased healthcare costs. This study characterizes phenotypic and genomic features of isolates from clinical samples in Karachi, Pakistan. Among 507 isolates, 213 (42%) were carbapenem-resistant based on disk diffusion and MIC testing. Urine (29.7%) and blood (28.3%) were the most common sources, with infections predominantly affecting males (64.7%) and individuals aged 50-70 years. Colistin was the only antibiotic showing consistent activity against these isolates. The whole-genome sequencing of 24 carbapenem-resistant (CR-KP) isolates revealed (45.8%) as the dominant carbapenemase gene, followed by (12.5%) and (54.2%). Other detected variants included , , , and . The predominant beta-lactamase gene was (91.6%), followed by , , and . Sequence types ST147, ST231, ST29, and ST11 were associated with resistance. Plasmid profiling revealed IncR (61.5%), IncL (15.4%), and IncC (7.7%) as common plasmid types. Importantly, resistance was driven not only by acquired genes but also by chromosomal mutations. Porin mutations in OmpK36 and OmpK37 (e.g., P170M, I128M, N230G, A217S) reduced drug influx, while and mutations (e.g., P161R, G164A, P157*) led to efflux pump overexpression, enhancing resistance to fluoroquinolones and tigecycline. These findings highlight a complex resistance landscape driven by diverse carbapenemases and ESBLs, underlining the urgent need for robust antimicrobial stewardship and surveillance strategies.