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Purpose This study investigated the clinical epidemiology, antimicrobial susceptibility, and molecular epidemiology of Corynebacterium striatum isolates. Patients and methods An observational study was conducted at a university hospital in the Republic of Korea from August to December 2016. All subjects were patients who tested positive for C. striatum clinically. Clinical data were analyzed to evaluate the microbiological and genotypic characteristics of C. striatum strains. Results Sixty-seven C. striatum isolates recovered from non-duplicated patients were characterized. Patients were classified into three groups according to the infection type: nosocomial infection (71.6%), health care-associated infection (8.7%), and community-acquired infection (18.8%). The most common clinical specimens were urine (35.8%) and skin abscesses (32.8%). Fifty-two (77.6%) isolates showed multidrug resistance, defined as resistance to ≥3 different antibiotic families. All strains were susceptible to vancomycin and linezolid. Resistance to other antibiotics varied: penicillin (n=65; 97.0%), ampicillin (n=63; 94.0%), cefotaxime (n=64; 95.5%), and levofloxacin (n=61; 91.0%). Phylogenetic analysis identified all 16 S rRNA gene sequences of the 67 isolates as those of C. striatum, where 98%-99% were homologous to C. striatum ATCC 6940. In multilocus sequence typing for internal transcribed spacer region, gyrA, and rpoB sequencing, the most predominant sequence types (STs) were ST2, ST3, ST6, and ST5. Conclusion C. striatum isolates may cause opportunistic infections associated with nosocomial infections through horizontal transmission. The presence of multidrug resistance and intra-hospital dissemination implicate C. striatum isolates as a potential target pathogen for infection control and antimicrobial stewardship programs.