Health care workers (HCWs) in high tuberculosis (TB) prevalence countries have to care for many cases, thus increasing their risk of infection. The objective of the study was to compare the prevalence of latent TB infection (LTBI) between general HCWs and TB HCWs, and also to explore the associated factors. A cross-sectional study was conducted in Nay Pyi Taw, Myanmar from September 2019 to January 2020. Staff working at two general hospitals were recruited. Those allocated for TB care were classified as TB HCWs, while the remaining were classified as general HCWs. Participants were interviewed using a structured questionnaire, and screened for LTBI using a tuberculin skin test (TST). Individuals who had an induration of 10 mm or more with normal chest radiograph were regarded as having LTBI. The prevalence of LTBI among general HCWs was 2.04 times higher than that of TB HCWs (31.2% vs. 15.3%, p < 0.001). The associated factors for LTBI included low education level, duration of work experience ≥ 10 years, a low knowledge of regular TB screening, and teaching cough etiquette to TB patients. The higher prevalence of LTBI in the general HCWs in this study was due to confounding by education and experience. After adjustment for these, we have no evidence to support that either group of HCWs had higher LTBI risk.