Aims: In November 2015, hepatitis C outbreak related with healthcare- associated infection was reported in Korea. To assess the exact cause of hepatitis C outbreak, we analyzed the sequences of HCV from plasma of patients and determined a correlation between HCV genotypes and transmission route. A retrospective survey was carried out to determine a possible nosocomial transmission. Methods: HCV RNA viral load test and genotyping was performed from the plasma of HCV Ab positive persons with the visit history in the clinic since May 2008. Subsequently, the Core-E2 and NS5B regions of the HCV from RNA positive sample patients were analyzed by the phylogenetic tree with maximum likelihood analysis. To identify risk factors for transmission and epidemiological relationship among the patients, we reviewed the patients’ medical records and assessed the state of infection control practice and the environmental inspection of the clinic. Results: Among the 1,721 tested persons, anti-HCV IgG positives were 101 cases and the 70 patients were the HCV RNA positive. Among the 61 patients whose HCV viral load was higher than detection limit, 41 (67.2%) were classified in genotype (GT) 1a, 1 (1.6%) in GT 1b, 18 (29.5%) in GT 1, and 1 (1.6%) in GT 2. 18 in GT 1 were further genotyped as 11 in GT 1a, 1 GT in 1b by sequence analysis. The sequences of core-E2 and NS5B regions of 45 patients were constructed as a monophyletic cluster apart from the previously reported GT 1a. Also sequences of HCV from the patients and environmental specimens were well matched after the sequence analysis in partial E1 gene region. By detecting HCV GT 1a RNA in environmental specimens, we proved this outbreak to be the health-care associated infection by reuse of syringes and contaminated multi-dose vials. Conclusions: We reported the first molecular epidemiological investigation of the HCV outbreak by a large cluster of rarely reported HCV GT 1a in Korea. The comprehensive analysis using molecular phylogenetics will play a key role in the investigation of infection sources in the outbreaks of hepatitis C virus and other pathogens.