To evaluate the risk of tuberculosis (TB) infection, the General Directorate of Epidemiology of the Ministry of Health developed a sentinel surveillance system in health centers located in the Constitutional Province of Callao. This surveillance system included the diagnosis of latent tuberculosis infection (LTI) using interferon gamma release assays. The objective of the present study was to estimate the prevalence of LTI in health workers in a region with a high burden of TB. The prevalence of LTI in health workers was 56%. The prevalence of LTI was 63% in workers with more than 10 years of service and 58–60% in workers with more than 35 years of service. The prevalence of LTI in health workers from primary health care centers was high, and longer service time was one of the main risk factors.