The objective of this prospective cross-sectional study was to compare a Mycobacterium tuberculosis-specific interferon gamma (IFN-γ) enzyme linked immunosorbent assay [QuantiFERON-TB Gold In-Tube (QFT-GIT)] test with tuberculin skin test (TST) for detection of latent tuberculosis infection (LTBI) in patients with juvenile idiopathic arthritis (JIA). To our knowledge, this is the first study evaluating the performance of QFT-GIT in comparison with TST in JIA. A cross-sectional study of 39 children with JIA and 40 healthy controls was conducted in İzmir, Turkey. Blood was for drawn for the QFT-GIT assay prior to administration of the TST using 5 tubercullin units (TU) of purified protein derivative (PPD-S). A positive TST was defined as ≥10 mm for JIA and ≥15 mm for controls. Statistical analysis was performed using SPSS version 16.0 for Windows. There were no significant differences between JIA patients and controls for age, sex, and Bacillus Calmette-Guérin (BCG) vaccination. Of patients, 70% had active JIA disease. The median TST induration was 5.8 mm (±5.7 mm) for JIA and 10.7 mm (±4.5 mm) for the control group, which was statistically significant (p = 0.000). The rate of patients who showed no reaction to TST was 38%, of which 93% had active disease. There were two patients who had positive IFN-γ results but negative TST, who had systemic and polyarticular type JIA, respectively. Overall agreement between TST and QFT-GIT was low both in JIA and control group (κ value =0.06 and 0.10, respectively). TST may be inadequate to diagnose LTBI in JIA patients. The IFN-γ assay may be useful to identify false negative TST response in cases with latent M. tuberculosis infection. The combination of IF QFT-GIT method with TST would provide successful diagnostic screening for LTBI in JIA, particularly prior to anti-tumor necrosis factor treatment. Long-term prospective studies are still necessary to appreciate the advantages and the applicability of these tests in pediatrics.