Overtaken by the coronavirus disease 2019 pandemic, human tuberculosis (TB) became currently the second leading cause of death worldwide due to a single infectious agent, Mycobacterium tuberculosis (Mtb). [1] Culture filtrate protein 32 (CFP32) is a culture filtrate antigen restricted to the Mtb complex. [2-4] It is associated with the virulence of mycobacterial strains and, thus, weakly expressed by the Bacillus Calmette-Guerin (BCG) vaccine strain. [4,5] The DNA-based booster vaccine based on CFP32 showed durable Th1 and strong humoral responses in mice. [6] In previous studies, we detected anti-CFP32 immunoglobulin (Ig) G in infected human sera, thus we developed a serological enzyme-linked immunosorbent assay (ELISA) using Pichia (P) pastoris recombinant CFP32 (rCFP32) antigen. [7,8] Interestingly, we showed that CFP32 exhibited higher immunoreactivity when produced in the yeast P. pastoris in comparison to Escherichia coli. [8] We showed that the rCFP32 produced in the yeast P. pastoris retains protein posttranslational events and homodimeric folding which may better mimic the protein's native conformation. [9] Background: We previously reported the development of an enzyme-linked immunosorbent assay for the detection of the immunoglobulin G (IgG) response to Mycobacterium tuberculosis virulence factor -culture filtrate protein 32 (CFP32). The assay achieved high performance in comparing healthy Bacillus Calmette-Guerin-vaccinated controls with active tuberculosis (TB) patients from the Tunisian population. Herein, we aimed to assess the anti-CFP32 IgG response in suspected or confirmed active pulmonary TB individuals in different endemic settings. Methods: Serum samples were obtained from 224 donors from African and Latin American countries with variable levels of TB endemicity and different ethnical origins. Receiver operating characteristic curve was used to evaluate the performance of the serological assay. Results: The area under the curve was 0.70. The use of a cutoff level of 0.65 gave 67% and 68% sensitivity and specificity, respectively, regardless of ethnicity and endemicity. Except for the suspected Latin American group, overall multiple comparisons of medians pointed out the stability of the anti-CFP32 IgG response across the different endemic settings. Therefore, endemicity and ethnicity seem not to affect anti-CFP32 IgG response, mainly for detecting confirmed active TB individuals. Conclusions: These findings suggest that the inclusion of CFP32 epitopes in multi-antigen TB assay could attenuate serological differences related to heterogeneous endemicity and ethnicity. For this purpose, we further identified B-cell epitopes belonging to CFP32 by an in silico analysis.