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Introduction Data are limited regarding tuberculosis (TB) and latent TB infection prevalence in Rwandan health facilities. Methods We conducted a cross-sectional survey among healthcare workers (HCWs) in Kigali during 2010. We purposively selected the public referral hospital, both district hospitals, and randomly selected 7 of 17 health centers. School workers (SWs) from the nearest willing public schools served as a local reference group. We tested for latent TB infection (LTBI) using tuberculin skin testing (TST) and asked about past TB disease. We assessed risk of LTBI and past history of TB disease associated with hospital employment. Among HCWs, we assessed risk associated with facility type (district hospital, referral hospital, health center), work setting (inpatient, outpatient), and occupation. Results Age, gender, and HIV status was similar between the enrolled 1,131 HCWs and 381 SWs. LTBI was more prevalent among HCWs (62%) than SWs (39%). Adjusted odds of a positive TST result were 2.71 (95% CI 2.01–3.67) times greater among HCWs than SWs. Among HCWs, there was no detectable difference between prevalence of LTBI according to facility type, work setting, or occupation. Conclusion HCWs are at greater risk of LTBI, regardless of facility type, work setting, or occupation. The current status of TB infection control practices should be evaluated in the entire workforce in all Rwandan healthcare facilities.