Current guidelines advocate screening hemodialysis patients for latent tuberculosis infection; however, the tuberculin skin test (TST) is believed to be insensitive in this population. This study compared the diagnostic utility of the TST with that of an IFN-gamma assay (T-SPOT.TB) and the clinical consensus of an expert physician panel. A total of 203 patients with ESRD were evaluated for latent tuberculosis infection with the TST, T-SPOT.TB test, and an expert physician panel. Test results were compared with respect to their association with established tuberculosis risk factors. Tuberculosis infection, as estimated by the tuberculin test, T-SPOT.TB test, and expert physician panel, was detected in 12.8%, 35.5, and 26.1 of patients respectively. Among patients with a history of active tuberculosis and radiographic markers of previous infection, 78.6 and 72.7% had positive T.SPOT.TB results, compared with 21.4 and 18.2% who had positive tuberculin tests. The physician panel unanimously declared infection in these two groups. On multivariate analysis, a positive T-SPOT.TB test was associated with a history of active tuberculosis, radiographic markers of previous infection, and birth in an endemic country, whereas a physician panel diagnosis also was associated with a history of previous tuberculosis contact. The TST is insensitive in hemodialysis patients and is not recommended to be used in isolation to diagnose latent tuberculosis infection. It is suggested that a combination of T-SPOT.TB testing and medical assessment may be the most accurate screening method.