Screening for latent tuberculosis infection (LTBI) with the Mantoux tuberculin skin test (TST) has many limitations including false-positive results due to Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination. Three hundred ninety adult inmates with normal screening chest radiographs in a county jail were evaluated for LTBI using TST and an ESAT-6/CFP-10 peptide-based enzyme-linked immunospot assay (T-SPOT.TB). LTBI prevalence rates were 19.0% and 8.5% by T-SPOT.TB and TST, respectively. Overall agreement between test results was 82.8% (κ = 0.29). Positive T-SPOT.TB results were significantly associated with increased age (odds ratio [OR], 1.04; 95% confidence interval [CI], 1.01 to 1.06) and intravenous drug use history (OR, 2.92; 95% CI, 1.36 to 6.27). Positive TST results were significantly associated with increased age (OR, 1.06; 95% CI, 1.02 to 1.09) and foreign birth (OR, 6.61; 95% CI, 1.98 to 22.01). Discordant covariates between the assay results included increased age (OR, 0.96; 95% CI, 0.94 to 0.99) and intravenous drug use history (OR, 0.41; 95% CI, 0.19 to 0.88). T-SPOT.TB reactivity is unaffected by prior BCG vaccination. T-SPOT.TB may be more sensitive than TST in diagnosing LTBI among a moderate risk population of inmates, particularly those with intravenous drug use history. Longitudinal studies are needed to assess the positive predictive value of T-SPOT.TB in identifying those most likely to convert to active disease in general populations as well as in high-risk subpopulations.