Background Immunologic tests such as the tuberculin skin test (TST) and QuantiFERON®-TB Gold In-Tube test (QFT-GIT) are designed to detect Mycobacterium tuberculosis infection, both latent M.Tuberculosis infection (LTBI) and infection manifesting as active tuberculosis disease (TB). These tests need high specificity to minimize unnecessary treatment and high sensitivity to allow maximum detection and prevention of TB. Methods Estimate QFT-GIT specificity, compare QFT-GIT and TST results, and assess factor associations with test discordance among U.S. Navy recruits. Results Among 792 subjects with completed TST and QFT-GIT, 42(5.3%) had TST indurations ≤10mm, 23(2.9%) had indurations ≤15mm, 14(1.8%) had positive QFT-GIT results, and 5 (0.6%) had indeterminate QFT-GITs. Of 787 subjects with completed TST and determinate QFT-GIT, 510(64.8%) were at low-risk for infection, 277(35.2%) were at increased risk, and none had TB. Among 510 subjects at low-risk (presumed not infected), estimated TST specificity using a 15mm cutoff, 99.0% (95%CI: 98.2-99.9%), and QFT-GIT specificity, 98.8% (95%CI: 97.9-99.8%), were not significantly different (p<0.99). Most discordance was among recruits at increased risk of infection, and most was TST-positive but QFT-GIT-negative discordance. Of 18 recruits with TST ≤15mm but QFT-GIT negative discordance, 14 (78%) were at increased risk. TB prevalence in country of birth was the strongest predictor of positive TST results, positive QFT-GIT results, and TST-positive but QFT-GIT-negative discordance. Reactivity to M. avium purified protein derivative (PPD) was associated with positive TST results and with TST-positive but QFT-GIT-negative discordance using a 10 mm cutoff, but not using a 15 mm cutoff or with QFT-GIT results. Conclusions M.Tuberculosis infection prevalence was low, with the vast majority of infection occurring in recruits with recognizable risks. QFT-GIT and TST specificities were high and not significantly different. Negative QFT-GIT results among subjects with TST induration ≤15 mm who were born in countries with high TB prevalence, raise concerns.