Objective. We compared a whole-blood interferon-γ release assay (QuantiFERON-TB Gold In-Tube test, hereafter “QFT-in tube test”) with a tuberculin skin test (TST) to determine which test more accurately identified latent Mycobacterium tuberculosis infection in healthcare staff. Methods. A total of 481 hospital staff members were recruited from 5 hospitals in Melbourne, Australia. They provided information about demographic variables and tuberculosis (TB) risk factors (ie, birth or travel in a country with a high prevalence of TB, working in an occupation likely to involve contact with M. tuberculosis or individuals with TB, or being a household contact of an individual with a proven case of pulmonary TB). The QFT-in tube test and the TST were administered in accordance with standardized protocols. Concordance between the test results and positive risk factors was analyzed using the к statistic, the McNemar test, and logistic regression. Results. A total of 358 participants had both a TST result and a QFT-in tube test result available for comparison. There were fewer positive QFT-in tube test results than positive TST results (6.7% vs. 33.0%; P < .001). Agreement between the tests was poor (71%; к = 0.16). A positive QFT-in tube test result was associated with birth in a country with a high prevalence of TB, the number of years an individual had lived in a country with a high prevalence of TB (ie, the effect of each additional year, treated as a continuous variable), and high-risk occupational contact. A positive TST result was associated with older age, receipt of bacille Calmette-Guérin (BCG) vaccination, and working in an occupation that involved patient contact. Receipt of BCG vaccination was most strongly associated with discordant results in instances in which the TST result was positive and the QFT-in tube test result was negative. Conclusion. In a population of healthcare staff with a low prevalence of TB and a significant rate of BCG vaccination, a positive QFT-in tube test result was associated with the presence of known risk factors for TB exposure, whereas a positive TST result was more strongly associated with a prior history of BCG vaccination.