Background Multidrug-resistant Acinetobacter baumannii is an important causal pathogen of healthcare-associated infections, and colistin-resistant strains have recently emerged owing to the increased use of colistin. Using next-generation sequencing (NGS), a single whole-genome sequencing (WGS) protocol can identify and type pathogens, analyze genetic relationships among different pathogens, predict pathogenic transmissions, and detect antibiotic resistance genes. However, only a few studies have applied NGS in studying the resistance mechanism and epidemiology of colistin-resistant A. baumannii. This study aimed to elucidate the resistance mechanism of colistin-resistant A. baumannii and analyze its molecular epidemiology through WGS. Materials and methods The subjects in this study were patients who visited a university hospital between 2014 and 2018. Thirty colistin-resistant strains with high minimum inhibitory concentrations were selected from various patient samples, and WGS was performed. Comparative genomic analysis was performed for the 27 colistin-resistant A. baumannii strains using a colistin-susceptible strain as the reference genome. Results The WGS analysis found no mutation for lpxA, lpxC, lpx D, pmrA, pmrB, and mcr1, the genes known to be associated with colistin resistance. Fifty-seven coding sequences (CDS) showed differences; they included 13 CDS with known names and functions that contained 21 genes. From the whole-genome multi-locus sequence typing (wgMLST) and single nucleotide polymorphism (SNP) analyses, two major clusters were found for the colistin-resistant A. baumannii strains. However, no differences were observed by the time of detection for each cluster, the samples, the pattern of antibiotic resistance, or the patient characteristics. In the conventional MLST following the Oxford scheme, the typing result showed ST1809, ST451, ST191, ST1837, and ST369 in the global clone 2 (GC2), without any relation with the results of wgMLST and SNP analyses. Conclusion Based on the findings of the resistance gene analysis through WGS and comparative genomic analysis, the potential genes associated with colistin-resistance or CDS were examined. Furthermore, the analysis of molecular epidemiology through WGS regarding colistin-resistant A. baumannii may prove helpful in preventing infection by multidrug-resistant bacteria and controlling healthcare-associated infections.