Background: We investigated the prevalence of latent tuberculosis infection (LTBI) among the health care workers (HCWs) and analyzed its risk factors in South Korea. Methods: A standard questionnaire regarding the baseline demographics and risk factors for LTBI was given to each participant and tuberculin skin test (TST), QuantiFERON-TB GOLD In-Tube (QFT-GIT) assay, and chest radiography were performed. Results: A total of 493 participants, 152 (30.8%) doctors and 341 (69.2%) nurses were enrolled in eight tertiary referral hospitals. The mean age of the subjects was 30.6 years old, and 383 (77.7%) were female. Of the 152 doctors, 63 (41.4%) and 36 (23.7%) were positive by TST and by QTF-GIT, respectively, and among the 341 nurses, 119 (34.9%) and 49 (14.4%) had positive TST and QFT-GIT results, respectively. Overall, the agreement between the two tests was 0.22 by the chance corrected proportional agreement rate (kappa coefficient) in 493 subjects. Experience of working in tuberculosis (TB)-related departments was significantly associated with positive LTBI test results by QFT-GIT assay, not by TST. In multivariate analysis, only age was independently associated with increased risk of a positive TST result, while age and experience of working in TB-related departments (odds ratio, 2.29; 95% confidence interval, 1.01-5.12) were independently associated with increased risk of a positive QFT-GIT result. Conclusion: A high prevalence of LTBI was found among South Korean HCWs. Considering the association between the experience of working in TB-related departments and high risk of LTBI, QFT-GIT may be a better diagnostic test for LTBI than TST in HCWs.