Introduction Anemia is a common condition at tuberculosis diagnosis, and there is evidence that its prevalence is higher in patients with tuberculosis than in those infected with Mycobacterium tuberculosis and healthy controls. Information about anemia during tuberculosis diagnosis is still scarce in the Brazilian population. The aim of this study was to describe the prevalence of anemia in patients with tuberculosis cared for at a referral center and its association with clinical forms of tuberculosis and other characteristics of these patients. Materials and methods This was a retrospective cross-sectional study of tuberculosis patients diagnosed from January 2015 to December 2018 at the Clinical Research Laboratory on Mycobacteria (LAPCLIN-TB) of Evandro Chagas National Institute of Infectious Diseases (INI)/Oswaldo Cruz Foundation (Fiocruz). A database of an ongoing cohort study underway at this service since 2000 provided the baseline information on tuberculosis cases extracted from a visit template. Exploratory and logistic regression analyses were performed to verify associations between anemia and demographic characteristics, socioeconomic status, clinical conditions, and laboratory results. Results Of the 328 cases reviewed, 70 were excluded, with258 retained. The prevalence of anemia was 61.2% (27.5% mild, 27.5% moderate and 6.2% severe). Among patients with anemia, 60.8% had normochromic normocytic anemia, and 27.8% showed hypochromic microcytic anemia. In logistic regression analysis, anemia was associated with a history of weight loss >10%, hospitalizations, coinfection with HIV, increased platelet count and microcytosis. Anemia was more frequent in the most severe clinical forms, such as meningeal and disseminated tuberculosis. Conclusions Anemia was highly prevalent in tuberculosis patients at diagnosis, predominantly as normochromic normocytic anemia and in mild and moderate forms. It was associated with baseline characteristics and conditions indicative of severe disease, suggesting that anemia could be a biomarker of tuberculosis severity.