Background <p>Multidrug-resistant <i>Acinetobacter</i> strains pose a significant threat to healthcare systems and have become a growing concern. A state-of-the-art understanding of this formidable pathogen—including its antibacterial resistance profile, virulence factors, genomic diversity, and evolutionary dynamics—is crucial for implementing effective responses during a pandemic.</p> Objective <p>To explore the genomic diversity, antimicrobial resistance (AMR) profile, virulence factors, and evolutionary trends of multidrug-resistant <i>Acinetobacter</i> isolates collected in South Korea from 2010 to 2022, we conducted a comprehensive in silico analysis of 74 complete genome sequences.</p> Methods <p>74 <i>Acinetobacter</i> complete genomes were annotated using Prokka (v1.14.6), and a pangenome was constructed using Roary (v3.13). The genomes were systematically analyzed for antibiotic resistance genes (ARGs), virulence factor genes (VFGs), mobile genetic elements (MGEs), CRISPR/cas, and prophage using various in silico tools. Multi-locus sequence typing (MLST) was conducted using the Pasteur scheme via the MLST 2.0 web server. To assess genetic diversity, eBURST and whole-genome SNP-based phylogenetic analyses were employed. We performed a targeted analysis of carbapenem-resistant <i>Acinetobacter</i> strains, investigating the relationship between prophages and CRISPR/Cas systems.</p> Result <p>Pan-genome analysis showed an open genome structure (α = 0.4921) in <i>Acinetobacter</i>, indicating ongoing genetic evolution. A total of 77 unique resistance genes, linked to six resistance mechanisms and 21 drug classes, were identified, including bla_OXA-23, efflux pumps (<i>AdeIJK</i>, <i>AdeFGH</i>), and carbapenemases. Additionally, 68 virulence factors associated with adherence, biofilm formation, iron uptake, immune evasion, and serum resistance etc. were prevalent. Mobile genetic elements, such as Tn6207, Tn6209, Tn2006, Tn2008, and ISAba1/blaOXA-23 combinations, were identified, suggesting mechanisms for the spread of resistance genes. Seventeen types of prophage were identified, and a low prevalence of CRISPR sequence implies susceptibility to phage predation. The ST2 genotype was dominant, and SNP-based phylogeny showed significant genomic diversity.</p> Conclusion <p>This study provides a comprehensive understanding of the resistance, virulence, and mobile genetic element profile of <i>Acinetobacter</i> isolates from South Korea, laying the groundwork for future antimicrobial resistance research and intervention strategies, especially the bacteriophage treatment.</p>